Construction Of CTPV Expression Vectors

Capripox disease is a serious infectious disease caused by infection of Capripoxvirus(CPV) and distributes all over the world. It is the most serious pox disease in animals and with typical characters of fever and caused pox eruption in all body. The genome of capripoxvirus is very large and has strong ability to express foreign genes. Only ruminant animal such as goat, sheep and bovin have the transmissibility in nature environment. It has no infectiosity to human and other mammals. So this virus is an ideal vector for recombinant live vaccine of some imporant infection diseases in ruminant animals. It has proved that the goat pox -chick embryo attenuated have good effection in immune protection. Thus, to construct goat pox virus live vaccine vector will have the good future.The aim of this study is to consrtucted newly GTPV expression vector by gene recombination, GTPV vaccine strain (G14-STV44-55) used as template.In this study, three possible nonessential regions during replication of CPV were selected ,WI1L or VVP11 used as the promoter controling expression of foreign gene, GPT as reporter gene, we constructed expression vector and got mutant strain of GTPV by recombinating with parent virus G14-STV44-55 .Plasmid PLSEG was digested to get GPT, which is controled by promoter VV7.5K .And then the GPT gene was cloned and sequenced , using as reporter gene in selecting recombinant virus. WI1L and WP11 were respectively conbined with promoter VV7.5K in opposite direction by overlap PCR., which used as controling and selecting system. Then this recombinated DNA section was cloned and sequenced.Selecting and cloning nonessential regions of replication are the key step during constructing. In this study, three nonessential regions TK,γ-IFNR and RR of replication of CPV were chosen according to previously studies. The primers were designed ,making sure nonessential regions have 800-1000bp side sequence on both sides. PCR amplification of the aim genes and their side sequences using G14-STV44-55 as template. Then the aim genes were cloned to pGM-T vector and sequenced. GPT-promoter complex sections were respectively inserted into ACC651, Pmel and Bglll of TK,γ-IFNR and RR three nonessential regions of replication. Finally, we got six expression vectors named PGM-TK-I1L-GPT, PGM-TK-P11-GPT , PGM-IFNR-P11-GPT, PGM-IFNR-I1L-GPT , PGM-RR-I1L-GPT, PGM-RR-P11-GPT.Testis of 3 to 5 month old Iamb was collected and primary cell was culture by trypsin digesting. The six expression vectors were transfected to cells by Liposome 2000 .which was infected with G14-STV44-55.Then cells were cultured in 37℃,5%CO₂ untill CPE and virus was collected . Using MPA(mycophenolic acid) and andaminopterin-induced syndrome as selecting regiments, recombinant clones that had cell pathological changes were selected and then identified byPCR. The positive recombinant virus were purified with picking plaque and accessed the heredity stability with consecutive passage cultured. Finally, gene recombination happened between vectors containγ-IFNR and parent virus. So we got two recombinant virus strains. In other words,γ-IFNR is the nonessential region of goat pox virus.In this study, goat pox virus expression vectors were first constructed in China. Also we firstly obtained the mutagenic strain of recombinant virus. These will make great progress in studying multivalent vaccines based on gene recombinant of goatpoxvirus.