The Prokaryotic Expression Of The P32 Protein And The Deleted Expression Vectors Construct Of The KLP2 Of Goatpox Virus Of Southern Xinjiang Strains

Capripox is the most serious of the virus caused by Capripoxvirus(CaPV),the OIE lists Capripox as one of the major animal infectious diseases that must be notified of the outbreak,and it is classified as the 1st animal disease in China.These diseases` infection rate and fatality rate are very high,and it have a strong impact on sheep husbandry.At 2010,outbreak of Capripox was many areas happened in Southern Xinjiang,it caused that 15 thousand sheep and goat died,morbidity and mortality were 34% and 65%.First,propagated fetal sheep skin cells then isolated viruses.(1)preparation of fetal sheep skin cells: first adopted healthy fetal sheep ewes from slaughterhouse-cattle and sheep,then added DMEM with 10%FBS,culture primary cell at 37? and 5% CO2 incubator after some steps by butchering,cleaning,digestion and dispersed cell,then studied fetal sheep skin primary cell culture characteristics with continuous passage culture.(2)Isolated culture of virus: got sick sheep skin which from sheeppox epidemic at 2010,after some steps by butchering,grinding then made into homogenate and brought supernatant into the fetal sheep skin cells,added penicillin-streptomycin,DMEM with 10%FBS,culture primary cell at 37?and 5% CO2 incubator,thawing and refreezing collected virus when cells appeared obvious CPE,Continue to blind 10 generation,next we extracted genomic DNA from every cell-culture medium with virus and then did PCR amplification.Next,the prokaryotic expression of the P32 protein of Goatpox virus: P32 protein was a membrane protein goatpox virus.In order to study the P32 gene which separate from the southern Xinjiang Capripoxvirus,through the augmentation of PCR,we received P32 gene and its membrane outer zone(P32mw)and hydrophilic region(P3228-20),and constructed expression vectors respectively(PET42b-P32 and PET42b-P32mw?pET28a-P32-20)and then proceed prokaryotic expression.The homology of the nucleotide sequence and geneticevolutionary relationships were predicted,and the sequence of amino acids and protein secondary structure were speculated.And then,constructed the deleted expression vectors construct of the KLP2 of Goatpox virus.First we cloned KLP2 gene which is 2300 bp long with flanking sequence into the pGEM-T,used Hind III cutting site about 1000 bp gene segment,after that we inserted fusion gene fraction which fused GFP as report gene and VV7.5 as promoter segments,constructed gene of lack of carrier vectors respectively named PGEM-KLP2-VV7.5-1GFP.The results showed:(1)After fetal sheep skin cells grew by static adherence,were bursiform or triangle at the beginning,with the increase of cell density,cells became elongated spindle and lined with tightly.Precancerous cells grew well,cell boundary were clear and tidy,cells could be represented about 4days.From seventh generation,cell growth cycle became shorter,fall off easily and had floating dead cells.cells need be represented about 3days even 2days.Cells were represented to 13 th generation in this study,though it can grew,but a lot of dead cells fall off rejected further research.(2)when vaccined virus,cell's lesions appeared after 48 h,and it became to 90% when 96 h,virus CPE were cell became thin and long,cell clearance bigger,or some of cell concentrated round.All the culture genome of extend virus,used P32 primers detection,and the results were positive.(3)The results of the prokaryotic of the P32 protein showed P32 gene that separate from the southern Xinjiang Capripoxvirus has larger mutation,compared with control group,it added 6 nucleotide in100-105 bp,coded two amino acids named K and N,total length had 975 bp,324 aa,as similar with Gansu2009.Transmembrane analysis evinced P32 protein extramembrane domain was1-282 aa and transmembrane domain was 283-305 aa,intramembrane domain was 306-324 aa.Secondary structure analyzed that the molecular N and transmembrane domain were obvious hydrophobic structure and weak antigen city,but extramembrane domain possessed strong hydrophilic and strong antigen city.Protein expression showed full-length P32 protein is almost no expression in Escherichia coli,extramembrane domain proteins could be expressed that existed in the form of inclusion body,but it had a low expression quantity,and difficult topurify.(4)After constructed gene of lack of carrier vectors respectively named PGEM-KLP2-VV7.5-1GFP,we cultured BHK-21 cell,transfect PGEM-KLP2-VV7.5-1GFP into BHK-21 cell,from the result,we can saw green fluorescence,it explained we constructed the deleted expression vectors construct of the KLP2 successful.The characteristics of the genetic makeup and protein expression are showed by research of the Goatpox virus southern Xinjiang strains,it laid a theoretical foundation in the respect that in order to deeply understand the P32 protein and further study the Goatpox virus.And lack of goatpox virus KLP2 gene expression vector of the completion of building work for goatpox virus genetic engineering subunit vaccine and missing KLP2 gene vaccine research provides important help.