| The infection rate of Simian T-Lymphotropic/Leukemia Virus type I (STLV-1) and Herpesvirus simiae (BV) in China monkey is very high. It not only affected the animal health and experiment results, but also was harmful to operators. At present, the diagnosis method of the two diseases are separating and cell culturing virus as antigen, it is harmful to operators and more expensive. In order to obtain more and harmless antigen, this study used Simian T-Lymphotropic/Leukemia Virus type I (STLV-1) gag chemical synthesis, Herpesvirus simiae (BV) gD gene which is part of the conservative fragment, then expressed it in E.coli, purified it by affinity chromatography, and established a STLV-1, BV of ELISA diagnostic method, at the same time, established a liquid chip technology about detecting two viruses antibodies together, This will lay the foundation for further experiments.We synthesized of STLV-1gag, BV gd gene fragment according to the sequence published on Genbank. Linked the purpose fragments into expression plasmid pET15b after digestion and was sequenced. It was transformed into the expression host of BL21 (DE3).After induced optimization conditions is confirmed, we detected the amount and activity of expression by SDS-PAGE and WB. Then operated fermentation (10 L) and purification of recombinant protein STLV-1 gag, BV gd. Recombinant protein used as antigen to detect the corresponding antibody in monkey serum, and compared with commercialization of diagnostic kit results. At the same time, recombinant protein STLV-1 gag, BV gd as a probe coupled to different colors (different #) fluorescent microbeads to detect the corresponding antibodies in rhesus monkey serum, and compared the test results with ELISA test results. The results are as follows:1. Got sequence fragments STLV-1 gag (1323bp), BV gd (1194bp) from pJ vector which is chemical synthesis by DNA2.0 Inc, and linked purpose fragment with the expression vector pET15b,so recombinant expression plasmid pET15b-STLV-1 gag, pET15b-BV gd had been constructed.2. Transformed the plasmid into the expression host rosetta2 DE3 plysS which was identified correctly. Detected the amount and expression of activity by SDS-PAGE, Western Blot after induced optimization conditions was confirmed. The results showed that STLV-1 gag, BV gd recombinant protein were expressed successfully in the Escherichia coli, STLV-1 gag recombinant protein existed bacteria in the form of soluble, BV gd recombinant protein in the form of inclusion bodies.3. Induced recombinant protein expression in 10L fermenter and purified recombinant protein STLV-1 gag, BV gd by Ni2+affinity chromatography in AKTA purification system.4. Used STLV-1 gag recombinant protein as antigen for detecting 1304 monkey sera from Guangxi, the positive rate was 3.76%; used BV gd recombinant protein as antigen for detecting 514 monkey sera, the positive rate was 29.96%; the positive rates respectively 2.68%, 21.60% by commercial diagnostic kits. The results showed that detection rate of recombinant protein significantly higher than commercial diagnostic kit (P <0.05). Therefore STLV-1 gag, BV gd can be used as a good substitute virus antigen. This will eliminate the dangers and difficulty of separating and culturing virus. At the same time, this will provide a cheap, rapid, accurate and effective ELISA method which can help to purify SPF monkey.5. Compared to ELISA and fluorescent microbeads flow test results, it proved that the fluorescent microbeads detecting monkey serum not only was more rapid than ELISA method, but also more sensitive and high than indirect ELISA method. It's established a more sensitive, convenient and rapid fluorescent microbeads diagnostic technique which can detect multiple targets simultaneously in a reaction system. It is a qualitative leap compared to the traditional methods of detection. |
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