The Detection Of Levels Of Follistatin-315 In The Human Serum By Enzyme-liked Immunosorbent Assay

There are two major species of follistatin (FS) in vivo, which are FS-288 consisting of 288 amino acid residues and FS-315 consisting of 315 amino acid residues and play important roles in inhibiting the secretion of FSH from anterior pituitary cells and specifically binding with activin. In this study, in order to investigate the effects of FS in the related diseases, we established two-site ELISA for detecting the FS-315 level in the serum of normal people and patients by using anti- FS-315 C-terminal polyclonal antibody and anti-FS N-terminal monoclonal antibody. The detail procedures are as follows. 1. Construction of FS expression vector of C-terminal peptides of human FS-315 (1) Preparation of cDNA encoding C-terminal peptides of human FS-315 protein: we extracted the total RNA from fresh human ovary cells and amplified the cDNA for C-terminal peptides of FS-315 by using Takara one-step RT-PCR kit, and recovered specific PCR products by electrophoresis onto a sliver of DE81 cellulose membrane.(2) TA cloning: we ligated the target gene fragment of FS-315C and the pGEM–T plasmid by TA cloning, after that we transformed the recombinant plasmid into the JM109 competent cells. We obtained the positive clone by blue-white color test.(3) Identification of the positive recombinant plasmid: we extracted the positive plasmid by means of alkaline lysis with SDS, identified the positive plasmid which contained target gene fragment, sequenced by the Applied Biosystems 100 Model 377 DNA sequence auto-analyser, and the sequence of FS-315C is identical with GeneBank, so the plasmid was named as pGEM-FS-315C. (4) Construction and identification of prokaryotic expression plasmid of pGEX-FS-315C: We inserted the target gene of pGEM-FS-315C into pGEX4T-1, a GST fusion protein expression vector, after using EcoRⅠand XholⅠto digest the plasmid, we transformated it into E.coli DH5αby electroporation and extracted the plasmid and identificated it by restriction enzyme digestion, the result was identical with that we had expected. After sequencing with the 5'—primer, we choosed the clone of pGEX-FS-315C with correct sequences .2. IPTG-induced expression of GST-FS-315C fusion protein and identification of the production by SDS-PAGE:(1) IPTG-induced expression and identification of GST-FS-315C fusion protein: expression plasmid for pGEM-FS-315C was transformed into E.coli BL21, induced the expression of protein with IPTG , and brake the bacteria by sonication, after centrifugation, collected the supernatant containing the secretive GST-FS fusion protein, At last we used 10% SDS-PAGE gel to identify the expression of the fusion protein, the expressive GST-FS fusion protein accounts for about 20% of the total bacterial proteins.(2) Purification of GST-FS-315C fusion protein: we purified the GST-FS fusion protein by Glutathione-Sepharose 4B affinity chromatography column according to its instruction, and the purified GST-FS fusion protein was purified by SDS-PAGE, then we obtained the recombinant GST-FS-315C.3. Establishment of the two-site ELISA for FS-315(1) Preparation of the anti-human FS-315C terminal polyclonal antibody: we immunized the New Zealand rabbits with the purified GST-FS-315C, and obtained the antiserum of FS, then precipitated the antiserum with saturation ammonium sulfate and then purified it by ProteinA affinity chromatography. Thus we got the polyclonal antibody of the C-terminal peptide of FS.(2) Measurement of human FS-315 by Two-site ELISA : we used the anti- human FS N-terminal monoclonal antibody to coat the 96-well plate, and the anti-FS-315 C terminal polyconal antibody as the testing antibody. After that we established two-site ELISA analysis. By using this method we analyzed immunoreactive FS (Ir-FS) in many biological materials, and found FS-315 in serum , follicular fluid, amniotic fluid of normal people and the supernatant of cultured small cell lung cancer .In this study we also used IRMA to analyze the level of FS in many samples mentioned above. We use...