| Techniques such as gene cloning, cell culture, protein analysis, enzyme-linked immunoassay (ELISA) were used to construct the fusion plasmid of inhibin and HBsAg gene. Rats were immunized with inhibin expressing plasmid to study the factors affecting inhibin gene immunization and its effect on the reproduction and reproductive endocrinology. The main content presents as follows:1 Construction and identification of fusion expressing plasmid of inhibin and HBsAg geneIn order to construct inhibin eukaryotic expressing plasmid fused with HBsAg gene, the secondary structure and physical and chemical characteristics of the expected recombinant inhibin protein were analyzed by use of Anthwin software to predict the epitope. S gene with enzyme site sequence of EcoR I and Nde I was amplified by PCR from pCMV-S; Inhibin (1-32) gene with enzyme site sequence of Nde I and HindIII was cut from pUI; S and inhibin (1-32) gene were directly inserted into pcDNA3.1(-) and the recombinant pCIS was identified by restriction endonuclease digestion and sequencing. Then plasmid DNA coated in liposome was transfected into HeLa cells and cells after 3 days of transfection and positive clone after selecting with G418 for two weeks were harvested. SDS-PAGE and ELISA was employed to detect the cell product. The results showed that in the secondary structure of recombinant inhibin, helix consists of 6%, sheet consists of 39%, and turn 8%, and coil 47%. The epitope of inhibin were probably at amino acid serial 225-258 regarding its hydrophobicity, antigenicity and hydrophilicity etc. Analysis of restriction endonuclease digestion and sequencing showed that the sequence of the insert was 793bp, which was identical to designed gene. The recombinant plasmid can be expressed in HeLa cell, and the fusion protein weight was about 29KD. The recombinant inhibin had immunologic activity of inhibin, it can competed with the synthetic peptides of inhibin a (1-26) to bind the antibody against inhibin. In conclusion, the fusion gene expression vector could be expressed in eukaryotic cell and the product had the immunologic activity of inhibin.2 Immunoreactive character and affecting factors of inhibin gene immunizationIn order to study the immunoreactive character of inhibin gene immunization and itsaffecting factors, two serial studies were conducted to examine the value of P/N of antibody against inhibin and the ratio of rats with positive antibody. Rats in sexual maturity were all immunized intramuscularly in the quadriceps muscle. Study 1: Four groups of rats were injected with 0μg, 15μg, 25 μ g or 40 μg inhibin α (1-32) recombinant expression plasmid pcINH coated in liposome. A booster was given without liposome 20 days later (n=10). Study 2: Five groups of rats were injected with 10 μ g, 50 μ g, 100 μg pCIS, 50μ g pcDNA3.1 or 100μL 0.85% saline. A booster was given 20 days later (n=18). 12 rats in every group were given two boosters 20 days apart (n=12). All rats were anaesthetized with bupivacaine hydrochloride 24 hours before the immunization. The results showed that in study 1, after twice immunizations of pcINH, 50%( 13/26) of rats were detected in positive antibody against inhibin. The average of the value of antibody P/N value of 40 u g immunization group were remarkably higher than that of 15 μ g and 25 μ g on day 20 after primary immunization and day 10 after booster. The increase of immunization dosage didn't promote the ratio of rats with positive antibody. In study 2, 38.9%(23/54) of rats were detected in positive antibody against inhibin after twice immunizations of pCIS, 55.6%(20/36) of rats were detected in positive antibody against inhibin after three times immunizations of pCIS. The increase of immunization dosage raised the level of antibody and the booster raised the ratio of rats with positive antibody and the antibody P/N of positive rats. Groups immunized with 50 μ g and 100 μ g pCIS got the same ratio of positive rats on day 10 after second booster. In conclusion, immunization with the inhib...
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