促伪狂犬病毒gD蛋白可溶性表达标签的筛选及融合蛋白生物学活性的检测

Screening of soluble expression tags for pseudorabies virus gD proteins and detection of biological activity of fusion proteins

为筛选有助于伪狂犬病病毒(PRV)gD蛋白可溶性表达的标签,并检测其融合蛋白的生物学活性,本研究通过PCR扩增PRV gD基因后,分别连接携带MBP、SUMO、NusA和GST 4个不同促溶标签的原核表达载体,构建重组质粒pET21b-MBP-gD、pET21b-SUMO-gD、pET21b-NusA-gD和pET21b-GST-gD。经双酶切和基因测序鉴定正确后分别转化大肠杆菌BL21(DE3),采用IPTG诱导后经SDS-PAGE检测各重组蛋白的表达,筛选可溶性表达效果最好的标签。结果显示,各重组质粒经酶切鉴定均获得852 bp的目的条带与各自的载体条带,均与预期相符,进一步测序结果显示插入基因序列与密码子优化后的PRV gD基因序列一致。SDS-PAGE检测结果显示,MBP标签融合gD蛋白的可溶性表达量最大,GST标签次之,SUMO标签和NusA标签融合的gD蛋白则均以包涵体形式表达。因此,选用重组MBP-gD蛋白(rMBP-gD)做后续鉴定。将rMBP-gD经Ni-NTA柱纯化后采用western blot和间接ELISA检测其反应原性;采用间接免疫荧光试验(IFA)检测该重组蛋白与PK15细胞的结合;将该重组蛋白与PRV-GFP共孵育PK15细胞后,经流式细胞术检测其竞争结合PK15细胞,从而抑制PRV的感染情况。结果显示,rMBP-gD具有反应原性,且可以结合PK15细胞,rMBP-gD蛋白可竞争结合PK15细胞抑制PRV的感染。上述结果首次证实,可溶性原核表达的rMBP-gD表达效果好,且具有生物学活性,本研究为PRV基因工程亚单位疫苗的后续研究奠定了基础。

This study aims to screen tags that enhance the soluble expression of pseudorabies virus(PRV)gD protein and to detect the biological activity of the resulting fusion proteins.Recombinant plasmids pET21b-MBP-gD,pET21b-SUMO-gD,pET21b-NusA-gD,and pET21b-GST-gD were constructed by PCR amplification of the PRV gD gene and linkage with four prokaryotic expression vectors,each carrying a different solubilization tag:MBP,SUMO,NusA,and GST.These plasmids were transformed into E.coli BL21(DE3).After induction,SDS-PAGE was used to detect the expression of each recombinant protein,and the tag yielding the highest soluble expression was identified.The results demonstrated that the MBP-tagged gD protein had the most significant soluble expression,followed by the GST-tagged protein.In contrast,the SUMO-and NusA-tagged proteins were expressed as insoluble inclusion bodies.Consequently,the rMBP-gD fusion protein was selected for further characterization.The rMBP-gD protein was purified using a Ni-NTA column,and its immunoreactivity was assessed by western blot and indirect ELISA.Indirect immunofluorescence assay(IFA)was used to evaluate its binding to PK15 cells.The CCK-8 assay was employed to determine the safe concentration of rMBP-gD on PK15 cells.Furthermore,PK15 cells were co-incubated with PRV-GFP,and flow cytometry was used to assess the competitive binding of rMBP-gD to PK15 cells and its potential to inhibit PRV infection.Western blot and indirect ELISA results confirmed the immunoreactivity of rMBP-gD.IFA results indicated that rMBP-gD could bind to PK15 cells.Flow cytometry results demonstrated that rMBP-gD competitively bound to PK15 cells,thereby inhibiting PRV infection.These findings indicate that the prokaryotically expressed rMBP-gD fusion protein retains biological activity.In conclusion,this study screened four different solubilization tags,successfully expressed a soluble PRV gD fusion protein,and preliminarily identified its biological activity.This work lays a foundation for further research on PRV genetically engineered subunit vaccines.