重组PCV3 Cap蛋白植物乳杆菌的构建与表达验证

Construction and expression verification of recombinant PCV3 Cap protein in Lactobacillus plantarum

构建3型猪圆环病毒(PCV3)Cap蛋白的重组植物乳杆菌并进行验证。通过PCR方法扩增含1320信号肽和DCpep优化的PCV3 Cap基因(PCV3D),使用无缝克隆技术连接到pSIP411载体中,电转入克隆宿主乳球菌NZ3900中,提取测序结果正确的重组表达质粒NZ3900:pSIP411-PCV3D再次电转入植物乳杆菌Lp12,制备重组菌并验证其表达。结果显示:成功扩增出目的条带为789 bp的经过修饰和优化的PCV3 Cap基因(PCV3D)片段,重组表达质粒电转入植物乳杆菌Lp12,成功构建重组植物乳杆菌Lp12:pSIP411-PCV3D。Western blot、流式细胞术等方法检测重组蛋白表达,获得阳性结果,阳性率为47.9%;免疫荧光进一步表明重组蛋白能够在细胞中表达。本试验成功构建了1株表达PCV3 Cap蛋白的植物乳杆菌,为PCV3口服候选疫苗的开发研究提供依据。

Recombinant Lactobacillus plantarum with type 3 porcine circovirus(PCV3)Cap protein was constructed and validated.The 1320 signal peptide and DCpep-optimized PCV3 Cap gene(PCV3 D)were amplified by PCR,ligated into the pSIP411 vector using the seamless cloning technique,and electroporated into the cloning host Lactococcus NZ3900 to extract the recombinant expression plasmid NZ3900:pSIP411-PCV3 D.It was again electroporated into Lactobacillus plantarum Lp12,and recombinant Lactobacillus plantarum were prepared and their expression was verified.A modified and optimized PCV3 Cap gene(PCV3 D)fragment with a target band of 789 bp was successfully amplified.The recombinant expression plasmid was electroporated into Lactobacillus plantarum Lp12,and recombinant Lactobacillus plantarum Lp12:pSIP411-PCV3 D was successfully constructed.Western blot,flow cytometry and other methods were used to detect the expression of recombinant protein,and the positive result was obtained.The positive rate was 47.9%.Immunofluorescence further indicated that the recombinant protein is capable of being expressed in cells.In this study,a strain of Lactobacillus plantarum expressing PCV3 Cap protein was successfully constructed,which provided a theoretical basis for the development of PCV3 oral vaccine candidate.