猪传染性胃肠炎病毒S蛋白在乳酸菌中的表达

Expression of Porcine Transmissible Gastroenteritis Coronavirus Spike Glycoprotein in Lactobacillus

目的:构建猪传染性胃肠炎病毒S蛋白的细胞内表达重组乳酸乳球菌,确定其最佳表达条件,为重组乳酸菌作为口服疫苗防治猪传染性胃肠炎奠定基础。方法:根据猪传染性胃肠炎病毒纤突(S)蛋白的全基因序列及表达载体质粒的基因融合特点,设计一对引物,进行PCR,获得含有TGEVS基因4个主要抗原位点的约2000bp目的片段,将其与表达载体质粒pNZ8048进行连接,通过电转化进入宿主菌乳酸乳球菌NZ9000细胞内,在乳链菌肽(Nisin)的诱导下进行表达,确定最佳表达条件;并通过SDS-PAGE进行检测和Western-blot分析表达蛋白活性。结果:成功获得了TGEVS蛋白在乳酸乳球菌细胞内的表达并且表达的蛋白具有TGE全病毒的抗原性。确定了乳酸乳球菌表达TGEVS蛋白的最佳表达条件为在以1ng/ml的乳链杆菌肽nisin诱导下,诱导后3h,重组蛋白表达效率达最高,重组蛋白约占菌体总蛋白含量的8.7%。结论:在乳酸乳球菌细胞内表达的重组TGEVS蛋白获得了理想表达,为进一步研制开发防治TGE口服疫苗提供物质基础。

Objective： Recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis coronavirus （TGEV） spike glycoprotein S was constructed and decided the optimum expression condition. It had established base for recombinant LactobaciUus as vaccine preventing and curing TGE. Methods： The gene of S Glycoprotein was cloned into a Lactococcus lactis vector pNZ8048. An appro：dmately 2000bps fragmemt of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in recombinant strain pNZ8048- S/NZ9000. The recombinant intracellular glycoprotein S was detected by SDS- PAGE and Western - blot after induced by 1ng/ml nisin. The effect of different inducing time and different concentration of the inducer on the expression product was detected. Results： The result indicated that the expressed product maintain antigenicity of TGEV as we expected. The recombinant glycoprotein S, the mount of which can be reached approximately 8.7% of the total bacteria protein evaluated by densitometric scanning, was detected by SDS - PAGE after induced by 1 ng/ml nisin. The expression efficiency achieved tiptop after 3 hours induced by 1 ng/ml nisin. Conclusion： The recombinant TGEV S protein was achieved expressed in Lactococcus lactis and had established base for developing oral vaccine preventing and curing TGE.