表达牛病毒性腹泻病毒E2蛋白的重组乳酸菌的构建及蛋白免疫原性分析

试验旨在构建能表达牛病毒性腹泻病毒（Bovine viral diarrhea virus，BVDV）E2抗原蛋白的重组乳酸乳球菌（Lactococcus lactis），为进一步研制BVDV乳酸菌口服活载体疫苗奠定基础。将BVDV E2基因克隆后测序，根据乳酸乳球菌的密码子偏嗜性进行优化，再将优化的基因片段插入表达载体pNZ8148中，并电转化乳酸乳球菌NZ9000感受态细胞，构建重组乳酸菌pNZ8148-E2/NZ9000，经1 ng/mL乳链菌肽诱导表达后，对菌体物进行了SDS-PAGE和Western blotting分析。将重组乳酸菌pNZ8148-E2/NZ9000口服免疫6~12月龄健康犊牛，在免疫后不同时间点采集血液样品并分离血清，用间接ELISA方法检测抗体水平。结果显示，PCR扩增到了1 149 bp的目的片段，乳酸菌密码子偏嗜性优化后，GC含量从45.28%变为34.30%。重组质粒pNZ8148-E2经酶切鉴定插入片段与预期大小相符，在菌体裂解物中出现大小约42 ku的条带，与预期蛋白大小一致，且该蛋白可与BVDV E2抗体反应。在免疫犊牛的血清中检测到特异性抗BVDV E2蛋白的抗体。本研究结果表明，表达BVDV E2蛋白的重组乳酸菌口服免疫可诱导犊牛产生特异性的体液免疫反应，该重组菌具有较好的免疫原性。

Construction of Recombinant Lactococcus Expressing E2 Protein of Bovine Viral Diarrhea Virus and Immunogenicity Analysis of the Protein

The aim of this study was to construct a recombinant Lactococcus lactis expressing E2 antigen protein of Bovine viral diarrhea virus (BVDV), and lay a foundation for further development of oral live vector vaccine of BVDV.The BVDV E2 gene was amplified and sequenced, the gene sequence was optimized and synthesized according to the codon bias of the Lactococcus lactis strain and inserted into the pNZ8148 expression vectors, the recombinant strain pNZ8148-E2/NZ9000 was constructed by electroporation of competent cells of Lactococcus lactis NZ9000, and the induced with 1 ng/mL Nisin, and E2 expression was analyzed by SDS-PAGE and Western blotting.Healthy calves aged 6-12 months were immunized orally with recombinant Lactobacillus pNZ8148-E2/NZ9000, and blood samples were collected and serum was separated at different time after immunization.The antibody level was detected by indirect ELISA.The results showed that the target fragment of 1 149 bp was amplified by PCR.After optimizing the codon preference of Lactococcus lactis, the GC content changed from 45.28% to 34.30%.The pNZ8148-E2 plasmid was identified as the expected size by enzyme digestion.Approximate 42 ku fusion protein was observed from the cell lysates of pNZ8148-E2/NZ9000 in Western blotting.Furthermore, specific anti-E2 IgG was detected in serum of immunized calves.The results suggested oral immunization with E2-expressing Lactobacillus could induce immune response, and the recombinant Lactobacillus had good immunogenicity.