Fused transcript of rearranged genes scca1 and scca2, dna sequence encoding its, expressing plasmid (variants)

FIELD: molecular biology, biochemistry, medicine, oncology.

SUBSTANCE: invention relates to DNA sequences found in analysis of mDNA from squamous carcinoma cellular lines of different origin wherein these DNA sequences represent transcripts from rearranged genes SCCA1 and SCCA2. Result of rearrangement is formation of fused gene consisting of exon 2-7 of gene SCCA1 and exon 8 of gene SCCA2, or exons 2-7 of gene SCCA2 and exon 8 of gene SCCA1. Prepared expressing vectors comprising above said combinations of exons of two genes provide synthesis of corresponding fused protein in host-cell. Proposed sequences of nucleic acids and genetic constructions based on thereof represent novel agents for diagnosis squamous carcinomas.

EFFECT: valuable biological and medicinal properties of transcripts.

8 cl, 9 dwg, 1 tbl, 5 ex

 

The SCOPE of the INVENTION

This invention relates to a transcript to obtain a fused protein, fused to the gene, detected in squamous cell carcinomas, the detection of rearrangement and monoclonal antibodies specific for SCCA1, SCCA1/A2, SCCA2/A1 and SCCA2.

BACKGROUND of the INVENTION

The squamous cell carcinoma antigen (SCCA) is a serological marker for squamous cell carcinoma (SCC) of the cervix, lung, head and neck, of the external female genitalia (vulva) and the esophagus [1, 2]. It was originally purified from THE complex-4 of squamous cell carcinoma of the cervix man, with a molecular mass of 42-48 kDa [1, 3]. This antigen consists of more than 10 proteins, and isoelectric focusing of this antigen revealed two subtractio, acidic (pI<6,25) and neutral (pI>6,25) isoforms [4]. The difference in molecular weight, possibly due to the modification [5].

Cloning of cDNA SCCA shows that it belongs to the family of inhibitors of serine proteases (Sabinov) [6]. The subsequent cloning of the genomic region on chromosome 18q21.3 revealed two tandemly arranged gene [7]. More telomeric, the original SCCA, was named SCCA1, while more centromeric was named SCCA2 (figure 1). They both contain eight exons, and the estimated boundaries of the intron-exon, splicing sites, initiating codons and codons termali Vlada identical. They are 98% identical at the level of nucleotides (figure 2) and 92% identical at the amino acid level (figure 3). The calculated value of pI shows that the neutral isoform is encoded SCCA1 and acidic isoforms SCCA2. It was found that alternative splanirovannaya variant mRNA of both genes leads to proteins that are 50 amino acids and 21 amino acid shorter [5].

People serpina mapped in one of two chromosomal clusters. PI6, PI9 and ELNAH2 mapped in R, while PI8, Bumpin, PAI2, SCCA1, SCCA2, Headpin and Maspin mapped in 18q21.3 (figure 1) [7-12]. It is assumed that these clusters arose by two independent micromosaic duplications and multiple rounds vnutripolostnyh duplications [9]. Often it was reported that the region of chromosome 18q is a district with a high frequency of rurangirwa [9, 13-16]. Targets and functions of Sechenov not been elucidated fully. The most that is known is that the primary function is the regulation of the proteolytic events associated with coagulation, fibrinolysis, apoptosis and inflammation, but it was reported and alternative features, such as the transport of hormones and the regulation of blood pressure [17-24].

Although SCCA1 and SCCA2 are almost identical, they differ in their loops reactive sites (figure 2 and 3). SCCA1 inhibits papyrologie cysteine proteinase-kata is Shin S, K and L [25, 26], whereas SCCA2 inhibits chymotrypsinogen serine protease-cathepsin G and himizu of mastocytes [27]. Research loop reactive site (RSL) SCCA1 show that RSL is essential for inhibition of cysteine proteases [28]. The variable part of the RSL dictates the specificity of the proteinase targets detected mutants exchange RSL SCCA1 and SCCA2 and single mutants [28, 29]. Perhaps serpina share RSL-dependent mechanism for inhibition of both serine and cysteine proteases.

The biological role of SCCA1 and SCCA2 are not fully elucidated. They are considered to be inhibitory serpine. These results suggest that SCCA1 involved in apoptosis, and its expression makes cancer cells resistant to several mechanisms death by inhibiting apoptosis [30]. The role of expression of SCCA2 in cancer cells is still unclear. In normal tissue antigen SCCA may play a specific role during the maturation of the epidermis [5].

Recent studies using discriminatory monoclonal antibodies and polymerase chain reaction (PCR) showed that both SCCA1 and SCCA2 are expressed in suprabasal layers of stratified squamous columnar epithelium of the tongue, tonsils, esophagus, cervix and vagina, Taurus, Kassala thymus, some parts of the skin and in the multilayer is Jindrisska epithelium of the upper respiratory tract (in which no gas exchange occurs) [31]. In squamous cell carcinomas of the lung and head and neck SCCA1 and SCCA2 coexpressed in moderately or well-differentiated tumors. In contrast to previous studies using non-discriminative antibodies, these results show that there was differential expression between SCCA1 and SCCA2 in normal and malignant tissue. Previous results showed that SCCA2 is detected only in the peripheral parts of the tumor [32]. This contradiction may be due to differences between immunohistochemical methods and specificnosti antibodies [31]. It was reported that false positive results are often caused by contamination with saliva or sweat during the procedure of analysis [1]. Cataltepe et al. assume that SCCA in saliva originate from the squamous cells that line the mucosal surfaces of the upper part of the digestive tract [31].

Usually SCCA1 and SCCA2 are detected in the cytoplasm of squamous cells [31], but not in blood flow [33]. The antigen, which appears in the serum of patients with SCC, may be due to overproduction SCCA tumor cells and their normal cycle (34). It was reported that SCCA, detected in serum using radioimmunoassay with antibodies or RT-PCR, is mainly SCCA2 [1, 35, 36], but other studies using PCR indicate n is then, both antigen can be amplified and detected in samples of patients [37].

The concentration of serum present in patients with SCC, correlate with clinical stage and with the degree of histological differentiation of the tumor (1). For cervical cancer, some studies show a correlation between the values of the preprocessing and clinical outcome [1, 38-43]. Studies also show a correlation between high levels of SCCA and tumor volume. Relapse or progressive disease could be detected for several months before clinical detection [39]. Similar results observed for squamous cell carcinoma of the lung, vulva, head and neck, and esophagus [1, 2, 44 and 45]. In all these studies measured the overall level of SCCA. Recently developed a new analysis sELISA using discriminatory antibodies for SCCA1 and SCCA2 [33].

The INVENTION

This invention relates to the transcripts fused protein, mRNA, different genes to ensure fused protein, and, in particular, it provides for the detection of the slit gene, consisting of parts of SCCA1 and SCCA2. These fused genes have now been found in cell lines SCC of different origin (cervical, lung and pharynx). The invention also provides methods for establishing specific immunological reagents in order to determine the surveillance/detection of these fused proteins.

One protein is defined by the following amino acid sequence

MNSLSEANTK FMFDLFQQFR KSKENNIFYS PISITSALGM VLLGAKDNTA QQIKKVLHFD QVTENTTGKA ATYHVDRSGN VHHQFQKLLTE FNKSTDAYE LKIANKLFGE KTYLFLQEYL DAIKKFYQTS VESVDFANAP EESRKKINSW VESQTNEKIK LIPEGNIGS NTTLVLVNAI YFKGQWEKKF NKEDTKEEKF WPNKNTYKSI QMMRQYTSFH FASLEDVQAK VLEIPYKGKD LSMIVLLPNE IDGLQKLEEK LTAEKLMEWT SLQNMRETCV DLHLPRFKME ESYDLKDTLR TMGMVNIFNG DADLSGMTWS HGLSVSKVLH KAFVEVTEEG VEAAAATAVV VVELSSPSTN EEFCCNHPFL FFIRQNKTNS ILFYGRFSSP

based on the DNA sequence

ATGAATTCAC TCAGTGAAGC CAACACCAAG TTCATGTTCG ACCTGTTCCA ACAGTTCAGA AAATCAAAAG AGAACAACAT CTTCTATTCC CCTATCAGCA TCACATCAGC ATTAGGGATG GTCCTCTTAG GAGCCAAAGA CAACACTGCA CAACAGATTA AGAAGGTTCT TCACTTTGAT CAAGTCACAG AGAACACCAC AGGAAAAGCT GCAACATATC ATGTTGATAG GTCAGGAAAT GTTCATCACC AGTTTCAAAA GCTTCTGACT GAATTCAACA AATCCACTGA TGCATATGAG CTGAAGATCG CCAACAAGCT CTTCGGAGAA AAAACGTATC TATTTTTACA GGAATATTTA GATGCCATCA AGAAATTTTA CCAGACCAGT GTGGAATCTG TTGATTTTGC AAATGCTCCA GAAGAAAGTC GAAAGAAGAT TAACTCCTGG GTGGAAAGTC AAACGAATGA AAAAATTAAA AACCTAATTC CTGAAGGTAA TATTGGCAGC AATACCACAT TGGTTCTTGT GAACGCAATC TATTTCAAAG GGCAGTGGGA GAAGAAATTT AATAAAGAAG ATACTAAAGA GGAAAAATTT TGGCCAAACA AGAATACATA CAAGTCCATA CAGATGATGA GGCAATACAC ATCTTTTCAT TTTGCCTCGC TGGAGGATGT ACAGGCCAAG GTCCTGGAAA TACCATACAA AGGCAAAGAT CTAAGCATGA TTGTGTTGCT GCCAAATGAA ATCGATGGTC TCCAGAAGCT TGAAGAGAAA CTCACTGCTG AGAAATTGAT GGAATGGACA AGTTTGCAGA ATATGAGAGA GACATGTGTC GATTTACACT TACCTCGGTT CAAAATGGAA GAGAGCTATG ACCTCAAGGA CACGTTGAGA ACCATGGGAA TGGTGAATAT CTTCAATGGG GATGCAGACC TCTCAGGCAT GACCTGGAGC CACGGTCTCT CAGTATCTAA AGTCCTACAC AAGGCCTTTG TGGAGGTCAC TGAGGAGGGA GTGGAAGCTG CAGCTGCCAC CGCTGTAGTA GTAGTCGAAT TATCATCTCC TTCAACTAAT GAAGAGTTCT GTTGTAATCA CCCTTTCCTA TTCTTCATAA GGCAAAATAA GACCAACAGC ATCCTCTTCT ATGGCAGATT CTCATCCCCA TAGATGCAAT TAGTGTGTCA CT

DESCRIPTION DISTINCTIVE OPTIONS

One fused gene (figure 4) was detected by sequencing cDNA from cell lines SCC.

Cell lineOrigin is giving SCCA1SCCA2
CaSkiCervixNormalA1/A2
C4ICervixNormalNormal

AEasyN.A.A1/A2
CaLu3EasyNormalnormal
SkMESEasyNormalnormal
RPMI2650ThroatN.A.A1/A2

In accordance with the switching sequence from SCCA1 to SCCA2 point break DNA must be in intron 7 (figure 2). This gene is, therefore, controlled by the promoter region of SCCA1, but to produce a protein with SCCA2-specificity.

Further research showed that the transcript fused protein is found in various sites in the formation of SCCA1/A2 or SCCA2/A1, i.e. different genes for these antigens provide crossover transcripts with the promotor region of one gene and loop reactive site of another gene.

This leads to the main invention transcript fused protein of two different genes having homology of nucleotides for men is our least 80% in a particular area, the area of the crossover reaction.

These fused genes cloned and stored in the form of a plasmid constructs and transformed in various strains of E. coli.

Plasmid pGEX6P-3 SCCA1/A2 containing the fused gene was deposited with the European Collection of Cell Cultures on 14 March 2001 under Deposit number ESAS 01031315.

Was derived protein, and research associate with the formation of the complex shows the binding of a substrate fused gene with Cathepsin G, but not Cathepsin L (figure 8).

Fused gene can be detected by southern blot analysis of tumor DNA (figure 9). Fused gene can also be detected by PCR analysis and cloning and sequencing of cDNA.

EXAMPLE 1

Cloning SCCA

1.1. PCR amplification

mRNA from cell lines Caski (cervix), C4-I (cervix), E (easy), CaLu3 (easy), SkMes (easy) and RPMI2650 (SIP) was obtained with the use of the kit for purification of mRNA QuickPrep Micro mRNA Purification kit (Pharmacia) and cDNA was obtained using the kit for cDNA synthesis first chain (Pharmacia). The DNA fragment size 1218 BP covering the coding sequence SCCA, was amplified using PCR in a 100 μl reaction mixture containing 10 mm Tris-HCl pH cent to 8.85, 25 mm KCl, 5 mm (NH4)2SO4, 2 mm MgSO4(Boehringer), 0.2 mm dNTP (Pharmacia), 10 μm SCCA 1-7F (DNA sequences for all primers are shown in tab is itzá 1), 10 μm SCCA 391-V, 2 μl of cDNA and 2.5 E Pwo polymerase (Boehringer). After denaturation of the samples for 5 minutes at 96°conducted in total 30 cycles, each comprising denaturation for 15 seconds at 96°C, annealing for 15 seconds at 60°C and elongation for 30 seconds at 72°C. the PCR reaction was completed with a final elongation for 10 minutes at 72°C.

1.2. Detection of SCCA1 and SCCA2

The presence of SCCA1 the PCR products were detected by cleavage with restriction enzyme SacII with getting two fragments of size 245 and 973 BP, respectively, or by SCCA1-specific PCR using primers SCCA 1-7F and SCCA1 323-V in a standard PCR reaction (75 mm Tris-HCl pH 8.8, 20 mm (NH4)2SO4, 0.01% tween-20, 2 mm MgCl2, 0.2 mm dNTP, 10 μm of each primer, matrix and 0.025 U/µl reaction Taq polymerase; after denaturation of the samples for 5 minutes at 96°conducted in total 30 cycles, each comprising denaturation for 15 seconds at 96°C, annealing for 15 seconds at the optimum temperature annealing and elongation for 30 seconds at 72°C. the PCR reaction was completed with a final elongation for 10 minutes at 72°C, TA=50°With obtaining fragment 997 P.N. Presence SCCA2 were detected standard PCR using SCCA 1-7F and SCCA2-specific primer, SCCA2 357-V,TA=60° With receipt of the fragment 1090 BP

1.3. Cloning

The PCR products were cloned using a kit for cloning PCR-Script Amp (Stratagene). Screening of colonies was performed using PCR, as described in 1.2 above. Plasmid DNA was obtained from selected clones containing SCCA1 or SCCA2, using the system for purification of DNA Wizard Plus Minipreps DNA Purification System (Promega).

1.4. DNA sequencing

Clones sequenced using sequencing using ABI Prism BigDye Terminator Cycle (PE Biosystems). The samples were subjected to electrophoresis on ABI Prism 310.

1.5. Re-cloning

The selected clones re-cloned in expressing vector pGEX-6P-3 (Pharmacia). Fragments cut out from the vector PCR-Script Amp using BamHI and XhoI and ligated in expressing the vector in the reaction of 10 μl, containing 1×ORA, 1 mm ATP, 50 ng of the split vector, insert SCCA, corresponding to the ratio of moles of ends of the vector to insert 1:5-1:8, and 7.5-10 E DNA ligase T4 (all from Pharmacia). The reaction tubes were incubated at 10°during the night and iactiveaware for 10 minutes at 65°C. 2-4 μl of the reaction was transformed into E. coli JM109 (46). Then plasmid DNA from selected clones were transformed into E. coli BL21 for protein expression.

1.6. Maintaining the cloned gene

Plasmid DNA (pGEX-6P-3, containing the fused gene SCCA1/A2) in 10 mm buffer solution of Tris-HCl pH 8.0, and stored at -80°C. DL is a renewed expression of the protein of plasmid DNA is transformed into competent E.coli BL21 according to Sambrook et al. (p.1.82-1.84 in reference 45). For more preferred plasmid DNA is transformed into E. coli JM109.

EXAMPLE 2

Expression and purification of protein

2.1. Expression of protein

Conditions expression was determined using drugs for the expression of a small scale. For large-scale expression cultures of 500 ml of 2×YT and 100 μg/ml ampicillin was inoculable 5 ml overnight culture and grown at 37°C. protein Expression was induced at OD600=0,5-1,3 adding IPTG to a final concentration of 0.1 mm. Culture, producing SSC1, grew within 4-16 hours, SCCA1/A2 for 16-18 hours. Culture, producing protein SCCA2, induced at OD600=1.2 to 1.4 and grown for 2-3 hours.

2.2. The protein purification

The cells were collected by centrifugation for 10 minutes at 2000 g, washed with 50 ml of TE pH 8.0 and was dissolved in 3 ml of TE/g of bacterial sludge. Added lysozyme to a final concentration of 800 μg/g sediment, and the mixture incubated on ice for 30-60 minutes and then frozen overnight at -70°C. was Added magnesium chloride and Tenkasu to a final concentration of 12 mm and 20 µg/g sediment, respectively. After incubation on ice for 30 minutes, samples were centrifuged within 30 minutes at 40,000 g. To each 0.5 ml of the supernatant was added to 50% Glutathione-Sepharose (Pharmacia), and incubated for 30 minutes 2 hours at room temperature with gentle shaking. The suspension was washed 5-7 times using 1×SFR. Protein GST-SCCA was suirable using 0.5-1 ml of reduced glutathione (Pharmacia), and incubated for 30-60 minutes at room temperature or over night at 4°With, in both cases with gentle shaking. Protein SCCA was suirable splitting between GST and SCCA. Added to 0.48 ml buffer for cleavage (50 mm Tris-HCl pH 7.0, 150 mm NaCl, 1 mm EDTA, 1 mm DTT) and 20 μl added before cutting protease, and samples were incubated at 4°with gentle shaking for 4 hours or over night. Proteins were analyzed using electrophoresis in LTO-PAG through Phast-system (Pharmacia).

2.3. The binding complex formation

Linking SCCA with the substrate with the formation of the complex was performed by mixing 2 μg SCCA-protein with 0.5 μg of Cathepsin G (Biodesign Int.) or 0.5 mg or 0.9 mg of Cathepsin L (Calbiochem) in 1×STR-buffer in a total volume of 4.5 ml. Samples were incubated at 37°C for 30 minutes. To each sample was added to 0.5 μl ber for the formation of the complex (20% LTOs, 140 mm mercaptoethanol, bromophenol blue). Samples were incubated for 3 minutes at 95°and analyzed by 12.5% of the LTO-PAG. Protein SCCA1/A2 forms a complex with Cathepsin G, but not Cathepsin L, which suggests that this protein is functional and has a substrate specificity which was mentioned SCCA2 (figure 8).

EXAMPLE 3

DNA analysis

3.1. The blot analysis for Southern

Approximately 10 μg of DNA obtained from cell lines SCC, as well as from blood samples of normal healthy volunteers, were digested with restrictase > PST or BamHI. Cleaved DNA was separated by 0.8% agarose, and transferred to membranes (Hybond N+, Pharmacia). Filters were prehybridization for 1 h and hybridized overnight at 60°With 20 ml of a solution containing 5×SSC, 0.1% of LTOs, 5% Textresult, liquid Liquid block block (Pharmacia)diluted 1:20, and DNA salmon sperm 100 µg/ml Concentration of the probe during hybridization was 10 ng/ml After hybridization the filters were washed in stringent conditions of a wash for 15 minutes in 1×SSC/0,1% LTOs and for 15 minutes in 0.2×SSC/1% LTOs, in both cases at 60°C. the Hybridization probe was detected using the detection module Gene Images CDP-Star (Pharmacia) with minor modifications. Filters were blocked for 1 hour at room temperature in a solution containing a liquid unit, diluted 1:7,5. Then they were incubated in buffer A (0.1 M Tris, 0.3 M NaCl, pH 9,5)/0.5 BSA for 15 minutes before adding the conjugate antiprogestin-HRP, diluted 1:6800, and then incubated for another 45 minutes. Filters were washed for 3×10 minutes in buffer And/0.3% tween-20 before adding the detecting reagent. The filters were incubated for 2 minutes, washed the short 2× SSC and wrapped in plastic film. Film Hyperfilm MP exhibited within 35 minutes.

3.2. Hybridization probes

Probes were received and were labeled using PCR in a reaction containing 60 μm each of dATP, dCTP and dGTP, 24 μm dTTP, 40 μm fluorescein-11-dUTP, 2 mm MgCl2, 3 microns direct primer, 3 μm reverse primer, 15 ng DNA template (SCCA2-containing plasmids), 1 E Taq polymerase and 1×STR-buffer (Advanced Biotechnologies). Probe I: fragment size 393 BP of exon 8 (nucleotides 802-1194), primers SCCA 266-273F and SCCA 391-V, TA=50°s; probe II: fragment of 126 BP of exon 8 (nucleotides 957-1082), primers SCCA2 319-324F and SCCA2 357-W, TA=50°s; probe III: fragment size 1194 BP covering the coding sequence and 22 nucleotide on the 3'end of this gene, the primers SCCA 1-7F and SCCA 391-V TA=60°C.

Blot the Southern split > PST DNA hybridizing with probe I, showed a different pattern of bands of DNA from SCC cell lines compared with the allocation of the bands normal control DNA (figure 9). DNA cleaved BamHI, also shows abnormal bands in comparison with normal control DNA.

3.3. PCR analysis

DNA extracted routine procedures of samples that may be analyzed using PCR using primers 7 and 8 (see table 1) in a standard PCR reaction, shows the product only in the samples containing the fused gene.

EXAMPLE 4

G is briday and monoclonal antibodies

4.1. Getting a hybrid and obtaining monoclonal antibodies reactive with SCCA1/A2, SCCA2 and SCCA1

Polyclonal antisera reactive with the antigen SCC received subcutaneous immunization of rabbits with recombinant antigen SCC and collecting immune serum, in accordance with standard procedures. The titer of the polyclonal antisera was tested by determining the reactivity of these antisera with biotinylating SCCA1/A2 and SCCA1, immobilized in tablets with streptavidin (Labsystems Oy, Helsinki, Finland), figure 6. Recombinant SCCA1/A2 and SCCA1 was biotinilated caproate ether Biotin-N-succinimide according to standard procedures.

Monoclonal antibodies reactive with SCCA1/A2 and SCCA2, was obtained by immunization of Balb/c mice intraperitoneally 10-50 µg of recombinant SCCA1/A2 in Freund Ribi. After immunization and booster 2-4 doses for 60-90 days, spleen cells from immunized mice were merged with myeloma cells P3 × 63Ag 8, as described [47].

Hybridoma producing antibodies reacting with SCCA1/A2 were selected using a screening ELISA supernatants hybrid in microtiter tablets coated with affinity-purified polyclonal anticorodal against mouse IgG+M (Jackson Measurement Res Lab, US). Then the wells were incubated with antigen SCCA1/A2 and after washing the bound antigen was detected by incubation with polyclonal cu is licinii antibodies against SCC and HRP-labeled swine antibody against rabbit Ig (Dako AS, Copenhagen, Denmark).

4.2. The reactivity of the selected hybrid with SCC antigen

The reactivity of the obtained hybridomas were tested in ELISA, similar to the ELISA screening procedure. Briefly, monoclonal antibodies produced by these hybridomas, was immobilized in microtiter tablets are coated with a polyclonal anticorodal against mouse IgG+M (Jackson Measurement Res Lab, US). Then the wells were incubated with 50 μl of different recombinant antigens SCC in SFR with 1% BSA for 1 hour after washing, the plates were incubated with 100 μl of rabbit antibodies against SCC, diluted 1/5000 in SFR-1% BSA, and incubated for 1 hour. Then connected the rabbit antibodies against SCC were detected by incubation with HRP-labeled swine antibody against rabbit Ig, and visualized using OPD substrate and determining the OD at 450 nm.

The figure 7 shows the reactivity of selected hybrid. SCC106, SCC114, SCC115 reacted only with SCCA1/A2, which indicates that they are specific to the fused protein SCCA1/A2. SCC100, SCC103 and SCC109 reacted with SCCA2 and SCCA1/A2, but not with SCCA1, suggesting that they are specific in respect of SCCA2. SCC110, SCC111 and SCC124 reacted with SCCA1 and SCCA1/A2, but not with SCCA2, suggesting that they are specific in relation to SCCA1.

SCC107, SCC119 and SCC128 reacted with all SCC-antigens, suggesting that they recognize a common epitope in SCA1 and SCCA2.

Cloned limiting dilution twice clones were produced antibodies reacting with SCCA1/A2, but negative in relation to SCCA1.

Monoclonal antibodies were obtained by cultivation in vitro hybridoma clones by inoculation of 104cells/ml in DMEM, 5% fetal calf serum in roller bottles and gave them to grow for 10-14 days. Then these monoclonal antibodies were purified from culture medium using Protein A (Bioprocessing Ltd, Durham, UK) - affinity chromatography in accordance with manufacturers ' recommendations.

EXAMPLE 5

Using the obtained monoclonal antibodies and recombinant proteins were able to develop immunoassays for specific definitions fused protein SCCA1/A2 and analyses specific to SCCA2 and SCCA1, respectively.

5.1. The immunoassays for determination of the fused protein SCCA1/A2

Analyses specific to the slit protein SCCA1/A2, but essentially negative in respect of SCCA1 and SCCA2 were created using antibodies among SCC106, SCC114 or SCC115 in combination with antibodies among SCC107, SCC119 or SCC128, see figure.

In the preferred form of analysis of antibody SCC107 used as catching antibody and SCC106 as the detecting antibody.

mAB SCC107 was biotinilated caproate ether NHRS, Sigma Chemical Co, US, using standard procedures, and is used as a capture antibody. mAB SCC106 conjugatively with HRP in accordance with the modification procedures Nakone.

Biotinylated mAB SCC107 and HRP-conjugated mAB SCC106 used in dvuhmetrovom EIA in accordance with the following Protocol.

Assay procedure

1. Add 50 µl of recombinant antigen SCCA (0-100 µg/l in STR, 60 g/l BSA, pH 7,2) + 100 ál of Biotin-mAB SCC107, 2 μg/ml in buffer for analysis in streptavidin coated microtiter tablets, Labsystems Oy, Helsinki, Finland.

2. Incubate for 1 hour±10 minutes with shaking.

3. Wash 3 times with 5 mm Tris buffer, 0.05% Tween 40, pH of 7.75.

4. Add 100 ál of HRP-mAB SCC106, 2 μg/ml in buffer for analysis.

5. Incubate for 1 hour±10 minutes with shaking.

6. Wash 6 times with 5 mm Tris buffer, 0.05% Tween 40, pH of 7.75.

7. Add 100 ál TMB ELISA Technology, US.

8. Incubate for 30 minutes ±5 minutes.

9. To determine the OD at 620 nm in an ELISA reader.

Curves dose-response to antigens SCCA1, SCCA2 and SCCA1/A2 revealed that this analysis was specific against recombinant antigen SCCA1/A2 cross-reactivity with SCCA1 or SCCA2 <5%.

5.2. Analyses for specific definitions SCCA2

Analyses specific to SCCA2, without significant reactivity with SCCA1/A2 and SCCA1 were created using antibodies among SCC100, SCC103 or SCC109 in combination with antibodies among SCC107, SCC119 or SCC128. In the preferred form of analysis of mAB SCC107 use is as a capture antibody and mAB SCC103 as the detecting antibody.

mAB SCC107 was biotinilated caproate ether NHRS, Sigma Chemical Co, US, using standard procedures, and used as a capture antibody. mAB SCC103 conjugatively with HRP type V (Sigma Chemical Co, US) in accordance with the modification procedures Nakone.

Biotinylated mAB SCC107 and HRP-conjugated mAB SCC103 used in dvuhmetrovom EIA in accordance with the following Protocol.

Assay procedure

1. Add 50 µl of recombinant antigen SCC (0-100 µg/l in STR, 60 g/l BSA, pH 7,2) + 100 ál of Biotin-mAB SCC107, 2 μg/ml in buffer for analysis in streptavidin coated microtiter tablets, Labsystems Oy, Helsinki, Finland.

2. Incubate for 1 hour ±10 minutes with shaking.

3. Wash 3 times with 5 mm Tris buffer, 0.05% Tween 40, pH of 7.75.

4. Add 100 ál of HRP-mAB SCC103, 2 μg/ml in buffer for analysis.

5. Incubate for 1 hour ±10 minutes with shaking.

6. Wash 6 times with 5 mm Tris buffer, 0.05% Tween 40, pH of 7.75.

7. Add 100 ál TMB ELISA Technology, US.

8. Incubate for 30 minutes ±5 minutes.

9. To determine the OD at 620 nm in an ELISA reader.

On the basis of the curves of the dose-response for slit proteins SCCA2, SCCA1 and SCCA1/A2 it was concluded that the analysis according to example 5.2 was specific in relation to SCCA2, with cross-reactivity against SCCA1 and SCCA1/A2 <5%.

5.3. Analyses for specific definitions SCCA

Analyses specific to SCCA1,without significant reactivity with SCCA2 and SCCA1/A2 were created using antibodies among SCC110, SCC111 or SCC124 in combination with antibodies among SCC107, SCC119 or SCC128. In a preferred configuration mAB SCC107 used as a capture antibody and mAB SCC124 as the detecting antibody.

mAB SCC107 was biotinilated caproate ether NHRS, Sigma Chemical Co, US, using standard procedures, and used as a capture antibody. mAB SCC124 conjugatively with HRP type V (Sigma Chemical Co, US) in accordance with the modification procedures Nakone.

Biotinylated mAB SCC107 and HRP-conjugated mAB SCC124 used in dvuhmetrovom EIA in accordance with the following Protocol.

Assay procedure

1. Add 50 ál of antigen SCC (0-100 µg/l in STR, 60 g/l BSA, pH 7,2) + 100 ál of Biotin-mAB SCC107, 2 μg/ml in buffer for analysis in streptavidin coated microtiter tablets, Labsystems Oy, Helsinki, Finland.

2. Incubate for 1 hour ±10 minutes with shaking.

3. Wash 3 times with 5 mm Tris buffer, 0.05% Tween 40, pH of 7.75.

4. Add 100 ál of HRP-mAB SCC124, 2 μg/ml in buffer for analysis.

5. Incubate for 1 hour ±10 minutes with shaking.

6. Wash 6 times with 5 mm Tris buffer, 0.05% Tween 40, pH of 7.75.

7. Add 100 ál TMB ELISA Technology, US.

8. Incubate for 30 minutes ±5 minutes.

9. To determine the OD at 620 nm in an ELISA reader.

On the basis of the antibodies in accordance with 5.3 can be created immunoassays specific for SCCA1, cross the reactivity against SCCA2 or SCCA1/A2 < 10%.

CAPTIONS TO FIGURES

1. Rurangirwa of chromosome 18. Cloning of the genomic region chromosome 18q21.3 identified two tandemly-located gene. Gene, located closer to the telomeric region of the original SCCA, was designated as SCCA1, whereas gene located closer to the centromeric region was identified SCCA2 (figure 1).

2. Comparison of the coding regions of DNA, exons 2-8 SCCA1 and SCCA2. Positions of introns are indicated as-Ix. The differences between the genes in bold. Italics region encoding loop reactive sites. Arrows indicate the locations of primers (table 1). Coding region identical at the nucleotide level is 98%.

3. Comparison of protein sequences SCCA1 and SCCA2. Positions of introns are indicated by dashed lines. The differences between the proteins are shown in bold. The blocks show the loop reactive sites. Proteins are identical at the amino acid level at 92%.

4. The nucleotide coding region DNA, exon 2-8 rearanging SCCA1/SCCA2. Sequences originating from SCCA1, shown in normal font, while the sequence originating from SCCA2 shown in bold. Positions of introns are indicated as-Ix. The differences between these genes are shown in gray. Italic indicates a region that encodes a reactive site loop.

5. Protein sequence if the CSOs protein SCCA1/SCCA2. Amino acids originating from SCCA1, shows the normal letters. Amino acids originating from SCCA2 shown in bold. Positions of introns are indicated by dashed lines. The differences between the proteins are shown in gray. The block shows the reactive site loop.

6. Title PABan regarding SCC antigen.

7. The reactivity of the obtained hybrid with different antigens SCC.

8. The analysis of binding with the formation of the complex fused protein SCCA1/A2. Track A: SCCA1/A2, track: SCCA1/A2, incubated with Cathepsin G, path: SCCA1/A2, incubated with Cathepsin L. the Complex SCCA1/A2 and Cathepsin G is shown by an arrow. Specified marker of molecular weight.

9. Southern blot analysis of genomic DNA cleaved > PST and hybridizing with probe I. Track A: RPMI2650 containing the fused gene SCCA1/SCCA2, track: normal DNA. Aberrant bands indicated by arrows. Specified marker of molecular weight.

1. Merged transcript to obtain a fused protein, encoded by exons 2-7 SCCA1 gene, fused with exon 8 of the gene SCCA2, or exons 2-7 of the gene SCCA2, fused with exon 8 of the gene SCCA1.

2. Merged the transcript according to claim 1, which is encoded by the nucleotide sequence shown in figure 4.

3. Merged the transcript according to claim 1, encoding the amino acid sequence shown in figure 5.

4. The DNA sequence corresponding to the nucleotide sequence of exons 2-7 SCCA1 gene, fused with a nucleotide sequence of exon 8 of the gene SCCA2, or the nucleotide sequence of exons 2-7 of the gene SCCA2, merged with the nucleotide sequence of exon 8 SCCA1 gene, the transcription of which is formed fused transcript according to claim 1.

5. The DNA sequence according to claim 4, where the DNA sequence corresponds to the nucleotide sequence of exons 2-7 SCCA1 gene, fused with a nucleotide sequence of exon 8 of the gene SCCA2, and shown in figure 4.

6. Expressing a plasmid containing the nucleotide sequence corresponding to exons 2-7 SCCA1 gene, fused with a nucleotide sequence of exon 8 of the gene SCCA2.

7. Expressing a plasmid containing the nucleotide sequence corresponding to exons 2-7 of the gene SCCA2, merged with the nucleotide sequence of exon 8 of the gene SCCA1.

8. Plasmid according to claim 6, containing the nucleotide sequence according to claim 5 and deposited in ESAS under Deposit number ESAS 01031315.



 

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