Fusion Expression And Identification Of MBP-GnRH-6

Objective The present study was to obtain GnRH vaccine with high quality and high throughput by using genetic engineering.Methods The synthetic mammalian GnRH-6 gene was inserted into the expression plasmid pMAL-c2x.The purified recombinant plasrnid was then transformed into E.coli BL21,and subsequently the expression of GnRH-6 gene was induced with IPTG.Afterwards,expressed protein products were purified by using amylose resin with affinity chromatograph method,and such purified products were analyzed by using SDS-PAGE.The reactiongenicity of fusion protein was identified by using indirect ELISA.In order to identify immunogenicity,the fusion protein was subjected to experimental animal followed by observing development and histological changes of mouse's testis.Results The GnRH-6 gene,fused with the gene encoding maltose binding protein,could be expressed at a high level in E.coli BL21.The affinity chromatograph using amylose resin was efficient in purifing the fusion protein(the concentration of the fusion protein is 4.06mg/mL).The molecular weight of the fusion protein was very similar to theoratic value(53.5kD).Moreover,the results of indirect ELISA indicate that the reactiongenicity of fusion protein to GnRH antibody was very high as evidenced by ratio of the OD₄₉₂of test group/the OD₄₉₂of the control group was larger than 2.1.The results of animal experiment showed that the length,width,girth and weight of the mouse's testis,after be immunized against fusion protein MBP-GnRH-6,were less than the control group.Significant difference of the length and weight was observed between the test and the control group(length:7.6233mm vs 8.1200mm,P＜0.01;weight: 0.09619g vs 0.1137g,P＜0.01).The girth in the test group was significantly less than that in the control group(20.0867mm vs 21.6322mm,0.01＜P＜0.05).The width of the testis was similar between two groups(5.4200mm vs 5.5911mm,P＞0.05).The slice of the testis obtained from mouse,immunized against fusion protein MBP-GnRH-6,showed significant histology changes regarding to sperrnatogenesis tubules,and arrangement of the spermatogonium,spermatocyte,and spermatid was not clear.In addition,the connective tissues were hyperplastic,and no sperm was produced in them.Although some testis had a few sperms,initiation of vacuole denaturalization was also observed in the spermatogenesis tubules.The structure of the spermatogenesis tubules in the control group was normal,and the spermatogonium,spermatocyte,spermatid and sperm were also normal besides their arrangement was clear.These results indicate that the fusion protein: MBP-GnRH-6,expressed by maltose binding protein fusion expression system pMAL-c2x, can stimulate the mice to produce GnRH antibody and make the testis atrophy.So the fusion protein MBP-GnRH-6 could be a candidate of immunocastration vaccine.