Construction Of Vectors Encodinging The Human S100C Gene And Preparation Of S100C Polyclonal Antibody

S100C is one member of the EF-hand S100 protein family, which is composed of calcium-binding regulatory proteins involving in cell cycle, cell differentiation, tumor growth, and extracellular matrix secretion. These proteins play an important role in biological activity of the body. The major function of S100C is on transduction of the calcium-dependent cell regulatory signals involved in cell proliferation, differentiation, apoptosis and the skeleton building. Recent studies have indicated that S100C may be closely associated with initiation, development and metastasis of tumor. Our recent studies using proteomics have demonstrated that S100C protein was abnormally expressed in lung cancer tissues, which suggested that this protein may play an important role in carcinogenesis of lung cancer.mRNA expression of S100C gene in lung cancer tissues was determined by semi-quantitative RT-PCR. S100C gene sequences were obtained by PCR method. Construction of S100C prokaryotic expression vector was carried out through design of suitable sites. Under optimized induction condition, the positive clones efficiently expressed fusion protein. Polyclonal antibodies were prepared using the fusion protein purified by Ni ion affinity chromatography in immunized rabbits. In the future studies, we will further examine the sensitivity and specificity in large volume of samples, and expect that this study will provide helpful information in studying other potential tumor markers.Methods1. Recombine the multiple cloning site of vector pET30a (+), the primers were designed by using Primer Premier 5.0 software. BamHI sites was introduced in the upstream primer, and XhoI-site was introduced in the downstream primer.2. mRNA expression level of S100C gene in lung cancer tissues and adjacent normal tissues inform 26 patients was determined by RT-PCR withβ-actin expression levels for internal standardization.. The full-length cDNA of S100C was cloned by PCR. The amplified DNA fragments were ligated into pET30a vector and then transformed into E.coli stain JM109. The positive clones were screened out.3. The positive clones were optimizated by adjusting induced concentration of IPTG and the induction time.4. Inclusion body was dissolved by using urea. The fusion proteins were purified by Ni ion affinity chromatography.5. Rabbits were immuuned with fusion protein together with Freund's adjuvant, and were immuuned thereafter every two weeks, with a total of 3 times.6. Antibody titers were detected by double Immunodiffusion, and antibody specificity was detected by using Western blotting. Results1. The expression level of S100C in lung cancer tissues was lower than that in adjacent tissue. The difference in S100C expression between the two groups was statistically significant (P<0.05).2. The prokaryotic expression vector of S100C was successfully constructed. The sequence homogenuity was up to 100%.3. The fusion protein could be expressed effeciently when induced by IPTG and the quantity was up to maxium when induced with 0.8 mmol/ L IPTG for 5h. The purity of fusion protein was 94.3%, and which was obtained by using the fusion protein of Ni ion affinity chromatography.4. High efficient and high specific polyclonal antibody was obtained by immunizing rabbits with fusion protein.Conclusions1. Ni Affinity chromatography is an effective method for purifying protein with His-Tag.2. Immunizing rabbits with purified S100C fusion protein can produce anti-S100C fusion protein antibody, which can react with human squamous tissue and adenoma tissue, thus lay foundation for detecting the specifity of the antibody with numerous samples in further research.