表达猪大肠杆菌987P-F18多表位肽重组乳酸乳球菌的构建及表征  

Construction and Characterization of Recombinant Lactococcus lactis Expressing Multi-epitope Peptide 987P-F18 of Porcine Escherichia coli

作　　者：张喜悦[1] 李昕 赵格[1] 赵建梅[1] 曲志娜[1] 刘俊辉[1] 王娟[1] 王珠琳 刘娜[1] 宋时萍 黄秀梅[1] 高玉斌 邹明 王君玮[1] Zhang Xiyue;Li Xin;Zhao Ge;Zhao Jianmei;Qu Zhina;Liu Junhui;Wang Juan;Wang Lin;Liu Na;Song Shiping;Huang Xiumei;Gao Yubin;Zou Ming;Wang Junwei(China Animal Health and Epidemiology Center,Qingdao 266032,Shandong,China;Qingdao Agricultural University,Qingdao 266000,Shandong,China)

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]青岛农业大学动物医学院,山东青岛266000

出　　处：《中国动物检疫》2024年第12期103-109,共7页China Animal Health Inspection

基　　金：国家重点研发计划项目(2022YFC2303900,2022YFD1301003);中国动物卫生与流行病学创新基金项目(DW2021016)。

摘　　要：为构建能表达猪大肠杆菌987P-F18多表位肽的重组乳酸乳球菌(Lactococcuslactis,L.lactis),以pET28a-9F1、pET28a-9F2、pET28a-9F3质粒作为模板,扩增具有不同表位排序的多表位肽序列,插入至表达载体pNZ8149中,并电转化至乳酸乳球菌NZ3900感受态细胞,分别构建重组乳酸乳球菌pNZ8149-9F1/L.lactis、pNZ8149-9F2/L.lactis和pNZ8149-9F3/L.lactis。经1ng/mL乳链菌肽诱导表达后,对菌体进行Westernblot分析,对菌株的稳定性进行测定,并绘制重组菌株与亲本菌株的生长曲线。结果显示:PCR扩增均获得了993bp的目的片段;构建的重组质粒经PCR、双酶切和测序鉴定,证实目标片段已被正确插入pNZ8149质粒;经Westerm blot分析,3株重组菌株裂解物在39.55kDa处出现可与987P抗体和F18抗体反应的目的条带,与预期蛋白大小一致;经20次传代后,将重组菌株接种于Eliker筛选培养基,发现98%以上的菌落仍为阳性菌落;与亲本菌株相比,外源蛋白的插入导致了重组菌株生长减缓,但无显著差异(P0.05)。结果表明,成功构建了3株表达猪大肠杆菌987P-F18多表位肽的重组乳酸乳球菌,其可稳定携带质粒,且生长性能并未受到显著影响。本试验构建的重组菌株为探索多表位肽重组乳酸乳球菌的最佳表位序列,研制有效的猪大肠杆菌987P-F18多表位肽重组乳酸乳球菌口服疫苗奠定了基础。In order to construct a recombinant Lactococcus lactis(L.lactis)expressing multi-epitope peptide 987PF18 of porcine Escherichia coli(E.coli),the plasmids of pET28a-9F1,pET28a-9F2,pET28a-9F3 were used as templates to amplify the sequences of multi-epitope peptide with different orders of epitopes,which were then inserted into expression vector pNZ8149,and were electrically transformed to L.lactis NZ3900 receptor cells to construct recombinant L.lactis strains of pNZ8149-9F1/L.lactis,pNZ8149-9F2/L.lactis and pNZ8149-9F3/L.lactis,respectively.The proteins were induced by nisin with the concentration of 1 ng/mL,and analyzed by Western blot.The stabilities of the strains were tested,and the growth curves of the recombinant strains and their parental strain were drawn.The results showed that target fragments with the length of 993 bp were amplified by PCR;the constructed recombinant plasmids were identified by PCR,double digestion and sequencing,confirming that the target fragments had been correctly inserted into the plasmid pNz8149;Western blot analysis showed that expected target bands of 39.55 kDa were observed,indicating that lysates of 3 recombinant strains could all react with antibodies against 987P and F18 protein respectively:the recombinant strains were inoculated in Eliker screening medium after being passaged for 20 times,and more than 98%colonies were still positive;compared to the parental strain,the recombinant strains grew slowly as caused by the insertion of exogenous proteins,but without significant difference(P>0.05).In conclusion,three strains of recombinant L.lactis expressing polyepitope peptide 987P-F18 of porcine E.coli were successfully constructed,which could carry plasmids stably,and their growth performances were not affected.A foundation was laid by the recombinant strains constructed in this experiment for exploring the optimal epitope sequence of recombinant L.lactis with multi-epitope peptides and developing the effective oral vaccines of L.lactis with multiepitope peptidesof E.coli 987

关 键 词：猪大肠杆菌 乳酸乳球菌 多表位 免疫原性 

分 类 号：S852.6[农业科学—基础兽医学]