Expression Of Early Proteins Of Capripoxvirus And Preliminary Research On Its Interactions

Capripoxvirus had occurred in many countries in the world. Once the disease’s outbreak occurred, always showed explosive popularity and caused huge economic losses of breeding industry, the import and export trade of sheep products would be impacted at the same time. In recent years, the foreign scholars found that several early proteins directly or indirectly affect the assembly of vaccinia virus,thus lead to normal virus particles can not be able to form the normal virion. It has yet not seen the reports about the assembly of capripoxvirus were affected by early proteins of capripoxvirus. The vaccinia virus is the mode virus of poxvirus family,therefore five proteins(L2R, A6 L, F10 L, A11 R, H7R) were expressed,and their interaction were researched through yeast two-hybrid of the study.In the first part of this study, I extracted the complete genome from the strain of Gulang capripoxvirus and the L2 R, A6 L, F10 L, A11 R, H7 R five genes were cloned by PCR amplification,then they were connected to PMD19-Simple vector and transformed into E. coli DH5ɑ competent,and the five plasmids were operated by BamHI + NotI endonuclease enzyme double digestion, then recycled the restriction enzyme fragment. Prokaryotic expression vectors were constructed after connection,and sequenced, then recombinant vectors were transformed into E. coli. Rosetta competent. The Western blot results showed that L2 R, A6 L, F10 L, H7 R four protein were expressed.In the second part of this study, L2 R, A6 L, F10 L, H7 R four genes were cloned and respectively connected to the yeast bait vector pGBKT7 and capture pGADT7 carrier. Later on, the plasmids were respectively transformed into yeast competent of Y2 HGold and Y787, after the bait vector self-activation and toxicity verification, processed the yeast mating. In the first screening, the mating product was screened by-Trp /-Leu double deficiency medium plates,-Trp /-Leu / X-A-Gal / Aba were used in the second round of screening,- Trp /-Leu /-His /-Ade / were used in the third round of screening,-Trp /-Leu /-His /-Ade /-X-A-Gal / Aba were used in the fouth round of screening。Results showed that the pGBKT7- F10 L had self-activation and only pGBKT7-A6 L and pGADT7-H7 R Hybrid had blue colonies in-Trp /-Leu /-His /-Ade /-X-A-Gal / Aba deficiency medium plates.In this study, five genes of early proteins were cloned and induced expression, in following tests, the interaction between A6 L and H7 R were improved by yeast two-hybrid. The study laid the foundation of the exploration of capripoxvirus assembly mechanism and the further development of capripoxvirus vaccine.