表达猪圆环病毒2型Cap蛋白重组链球菌的构建及其免疫原性分析  

Construction and immunogenicity analysis of recombinant Streptococcus expressing porcine circovirus type 2 Cap protein

作　　者：宗雪晴 赵贤杰 胡俊冶 余肇锋 李国潮 何颍欣 黄云飞 马春全[1,2] 李舜 李亚娟 付强 ZONG Xue-qing;ZHAO Xian-jie;HU Jun-ye;YU Zhao-feng;LI Guo-chao;HE Ying-xin;HUANG Yun-fei;MA Chun-quan;LI Shun;LI Ya-juan;FU Qiang(Department of Animal Medicine,School of Life Science and Engineering,Foshan University,Foshan 528225,China;Foshan University Veterinary Teaching Hospital,Foshan 528225,China)

机构地区：[1]佛山科学技术学院动物医学系,广东佛山528225 [2]佛山佛科院动物医院有限公司,广东佛山528225

出　　处：《中国预防兽医学报》2023年第6期611-620,共10页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31872443);广东省基础与应用基础研究基金项目(2022A1515140052);广东省重点建设学科科研能力提升项目(2022ZDJS036);大学生创新创业训练计划项目(202111847019)。

摘　　要：猪圆环病毒2型(PCV2)常与马链球菌兽疫亚种(SEZ)混合感染。为构建表达PCV2 Cap蛋白的重组SEZ并分析其免疫原性,本研究通过PCR扩增PCV2 Cap基因和SEZ疫苗株ST171 Szp基因的上下游同源臂基因(Szp-up、Szp-down),将上述PCR产物分别与pMD19-T载体连接,分别构建重组质粒pMD19-T-Cap、pMD19-T-Szp-up和pMD19-T-Szp-down。将pMD19-T-Szp-up与pG+host5分别双酶切后构建重组质粒pG+host5-Szp-up;将pMD19-T-Szp-down与pG+host5-Szp-up分别经双酶切后构建重组质粒pG+host5-Szp。上述重组质粒均经酶切和测序鉴定正确后分别经BamH I酶切pG+host5-Szp载体和pMD19-T-Cap载体后构建重组载体pG+host5-PCV2-Cap-Szp,并使PCV2 Cap基因替换Szp基因的166 bp~783 bp。经酶切和测序鉴定正确后将该质粒电转化ST171感受态细胞,通过红霉素抗性板筛选重组SEZ PCV2-Cap-Szp/ST171株。通过菌落计数分别绘制PCV2-Cap-Szp/ST171及其亲本菌株ST171的生长曲线;分别采用荧光定量PCR(qPCR)检测重组菌株Cap基因以及亲本菌株ST171 Szp基因的转录水平;通过液相色谱-串联质谱(LC-MS/MS)鉴定重组菌株Cap蛋白的表达。结果显示,亲本菌株和重组菌株的生长曲线基本一致,重组菌株中Cap基因的转录水平与亲本菌株中Szp基因的转录水平相当,且其中的蛋白肽段与PCV2 Cap蛋白的肽段相符,表明插入Szp基因不影响ST171的生长特性,且重组菌中的Cap基因可以正常转录并能够表达Cap蛋白。分别经小鼠腹腔接种不同剂量的重组菌株及亲本菌株,并采用改良寇氏法(Karber氏法)计算两株菌对小鼠的半数致死量(LD50)。结果显示,重组菌株及亲本菌株对小鼠的LD50分别为3.21×108 cfu/mL及2.0×10^(7) cfu/mL,虽然前者对小鼠的LD50有所升高,但其对小鼠的致病性并未增强。将重组菌株及亲本菌株灭活乳化后与商品化的PCV2灭活疫苗分别免疫小鼠,0.5 mL/只,二免后14 d采用间接ELISA检测各组小鼠血清中的Cap蛋白抗体水平,结果Porcine circovirus type 2(PCV2)is often co-infected with Streptococcus equi ssp.zooepidemicus(SEZ).To construct the recombinant SEZ expressing PCV2 Cap protein and analyze its immunogenicity,the upstream and downstream homologous arm gene sequences(Szp-up and Szp-down)of SEZ vaccine strain ST171 Szp gene and PCV2 Cap gene were amplified by PCR,respectively,and the PCR products were inserted into pMD19-T vector,resulting three recombinant plasmids pMD19-T-Cap,pMD19-T-Szp-up and pMD19-T-Szp-down.The recombinant plasmid pG+host5-Szp-up was constructed after double digestion of pMD19-T-Szp-up and pG+host5,respectively.The recombinant plasmid pG+host5-Szp was constructed after double digestion of pMD19-T-Szp-down and pG+host5-Szp-up,respectively.Identified by enzyme digestion and sequencing,the recombinant plasmids pG+host5-Szp vector and pMD19-T-Cap vector were then digested by BamH enzyme,respectively,to construct the recombinant vector pG+host5-PCV2-Cap-Szp.PCV2 Cap gene was integrated into the 166bp-783bp of Szp gene.The recombinant plasmid was electroconverted into ST171 competent cells,and the recombinant SEZ PCV2-Cap-Szp/ST171 strain was screened by erythromycin resistance plate.The growth curves of PCV2-Cap-Szp/ST171 and its parent strain ST171 were plotted by colony count.qPCR was used to detect the transcription levels of Cap gene of the recombinant strain and Szp gene of parent strain ST171.The expression of Cap protein was identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The results showed that the growth curves of the parent strain and the recombinant strain were basically the same.The transcription level of Cap gene in the recombinant strain was similar to that of Szp gene in the parent strain,and the protein peptide segment in the recombinant strain was consistent with the peptide segment of PCV2 Cap protein in the database,indicating that the insertion of Szp gene did not affect the growth characteristics of ST171.The Cap gene in the recombinant bacteria could be transcribed norma

关 键 词：猪圆环病毒2型 CAP蛋白 马链球菌兽疫亚种 重组菌株 

分 类 号：S852.6[农业科学—基础兽医学]