Construction Of Recombinant Virus Containing Antigen Prote In Of Mycoplasma Pneumoniae And Capripox Virus

Mycoplasma capris pneumonia is a respiratory system disease caused by Mycoplasma capris.The main pathogens are Mycoplasma capris subspecies,Mycoplasma capris subspecies,and Mycoplasma capris,which cause sepsis in goats,contagious pleuropneumonia in goats,and Mycoplasma capris pneumonia in sheep.For the prevention and control of Mycoplasma pneumoniae in sheep,China currently mainly uses attenuated and inactivated vaccines for prevention and control.However,although inactivated vaccines have certain immune protection effects,the immunization period is short and the labor cost of vaccination is high.Therefore,there is an urgent need for a new vaccine to prevent and control Mycoplasma pneumoniae in sheep.The sheep pox virus can insert foreign genes of 2645 kb without affecting virus reproduction and passage.It has the potential to develop recombinant multivalent vaccines,and the attenuated sheep pox virus vaccine has good immune effects,making it a necessary vaccine for immunization in the sheep industry.This study aims to construct a recombinant multivalent vaccine using the sheep pox virus vaccine stran as the vector and recombining the important antigen gene of Mycoplasma ovipneumoniae,in order to have immunoprotective effects on both sheep pox and Mycoplasma ovipneumoniae simultaneously.Firstly,a prokaryotic expression vector was constructed using the synthesized M17 gene of Mycoplasma capris subspecies goat pneumonia(accession number: WP＿024070888.1),the genetic evolution of the gene sequence was analyzed,and the structure of the encoded protein was predicted.The protein expression and identification of the M17 gene were performed using IPTG induction method.Secondly the M17 gene was amplified by PCR,digested by Nhe Ⅰ and Bss H II,and inserted into the constructed sheep pox virus expression vector pGM-TK13-P11-MSC-loxp-gpt-VV7.5-VV7.5-GFP to construct the multivalent recombinant plasmid of M17 sheeppox virus.The recombinant plasmid was transfected intosheep testicular cells infected with sheeppox virus vaccine strain GTPV AV41 by Lipofectamine2000,observe and select the fluorescent virus under a fluorescence microscope.After repeated freezethaw cycles,dilute and inoculate the primary cells of sheep testicles with a ratio of 10 times.Then select the fluorescent virus and dilute and purify it for 5 rounds.Results the prokaryotic expression vector M17-pET42 b of M17 was successfully constructed,and it was found that the size of M17 was 927 bp.After homologous comparison,the M17 gene sequence of goat mycoplasma MCCP1601 strain was closely related to the refere nce strain Mccp-JCVI-Syn3B-PSC09-L2(accession number:CP069346.1).Both of them were in the same branch with goat mycoplasma pneumonia subspecies,and the homology was as high as 94.8%-98.3%.Using bioinformatics technology to predict its protein structure,309 amino acids were encoded,and it was found that the secondary and tertiary structures were mainly composed of α Mainly spiral and irregular curls.SDS-PAGE analysis showed that M17 protein was largely expressed in precipitation,and the band was clear at34.2 KDa.Westernblot results showed that M17 protein was fused with His tag.The multivalent expression vector pGM-TK13-P11-M17-loxp-gpt-VV7.5-VV7.5-GFP of goat pox virus was successfully constructed and the recombinant virus was transfected into the cells,the recombinant virus obtained from transfected cells was purified through 5 rounds of screening to obtain a recombinant goat pneumonia antigen protein M17-sheep pox vaccinevirus.To screen highly conserved and immunogenic antigens as candidate strains for recombinant vaccines and lay a theoretical foundation for constructing multivalent recombinant vaccines.Lastly,bioinformatics technology and homologous recombination method were used to explore the biological function of heat shock HSP70/ IFN-γ/ TU gene of Mycoplasma pneumoniae antigen protein.The gene sequence was analyzed by homology alignment and protein structure prediction,and the triple gene was synthesized and digested by Nhe Ⅰ and Bss H Ⅱ.The triplet gene was ligated with the constructed sheep pox virus expression vector pGM-TK13-VV7.5-MSC-loxp-gpt-VV7.5-VV7.5-GFP to construct the multivalent recombinant plasmid of sheep mycoplasma triad gene-sheep pox virus.The recombinant plasmid was transfected into sheep testicular cells infected with sheep pox virus vaccine strain GTPV AV41 by Lipofectamine2000.The fluorescent virus was observed and picked under fluorescence microscope,the virus was frozen and thawed repeatedly.The fluorescent virus was selected by10-fold dilution and inoculated with sheep testicular primary cells,and then diluted and purified.Results the expression vector pGM-TK13-VV7.5-HSP70/IFN-γ/ TU-loxp-gpt-VV7.5-VV7.5-GFP of sheep mycoplasma triad gene-sheep pox virus was successfully constructed,and the size of2394 bp was found by sequencing.After homologous comparison,the heat shock protein HSP70 of Mycoplasma hyopneumoniae and the reference strain of Mycoplasma hyopneumoniae CVCC-384 are in the same branch,but have similar phylogenetic relationships with the reference strain of Mycoplasma hyopneumoniae 7448,with a high homology of up to 97.6%-99.2%.Using bioinformatics technology to predict its protein structure,310 amino acids were encoded,and it was found that the secondary and tertiary structures were mainly composed of α Mainly spiral and irregular curls.Successfully constructed the expression vector of the triple gene of sheep mycoplasma sheep pox virus,transfected the recombinant virus obtained from cells,and continued to purify the screened recombinant virus.