非洲猪瘟病毒B318L蛋白的多克隆抗体的制备及应用

Prokaryotic expression of African swine fever virus B318L protein and preparation of polyclonal antibody

非洲猪瘟病毒(ASFV)B318L是ASFV编码的晚期蛋白,具有香叶基香叶基合成酶活性。为了制备B318L的多克隆抗体并将其初步应用,本研究利用PCR方法从重组质粒p CAGGS-HA-B318L中扩增ASFV B318L基因构建重组表达质粒p GEX-6p-1-B318L,经PCR和测序鉴定正确后转化大肠杆菌BL21(DE3)经IPTG诱导并克隆至p GEX-6p-1载体中,采用GST亲和层析柱纯化重组蛋白,利用SDS-PAGE检测该蛋白的表达,采用western blot鉴定重组蛋白的纯化效果和反应原性,采用超微量紫外分光光度计测定蛋白浓度。结果显示,在约60 ku处出现特异性条带,且重组B318L蛋白(rB318L)主要以可溶性形式表达;纯化后在约60 ku处出现了单一特异性条带,经超微量紫外分光光度计测定蛋白浓度为10 mg/m L。将纯化的r B318L 4次免疫小鼠并于3免后一周采血,采用western blot鉴定该多克隆抗体(pAb),经Protein G亲和层析介质纯化后采用SDS-PAGE进一步鉴定纯化效果,采用间接ELISA检测p Ab的效价。结果显示,制备的小鼠血清(pAb)在60 ku处出现特异性条带,其与原核表达的r B318L发生了反应;该p Ab包含重、轻链且纯度较好,且其效价可达1∶32000。将不同剂量的p CAGGS-HA-B318L(1μg、2μg、4μg)分别转染HEK293T细胞,24 h后收集细胞,将MOI 1的ASFV HLJ/18株感染猪肺泡巨噬细胞(PAM),并分别于感染后6 h、12 h、24 h收集细胞。上述细胞均经相应处理后分别采用western blot、间接免疫荧光试验(IFA)(pCAGGS-HA-B318L转染剂量为1μg)检测制备的p Ab的反应原性。Western blot结果显示,HEK293T细胞中出现约35 ku的特异性条带,并随转染质粒浓度的增加B318L蛋白表达量增多;ASFV感染24 h后的PAM在35 ku处出现特异性条带,其余时间点均无该条带。IFA结果显示,过表达B318L蛋白的HEK293T细胞中出现红色荧光;ASFV感染12 h后的PAM中出现红色荧光,24 h后PAM中红色荧光的量增加。将制备的B318L p Ab用于免疫共沉淀(Co-IP)试验检测293T细胞中过表达的B318L、ASFV感染PAM后内源表达的B318L,以及两种细胞中表达的B38L蛋白与p Ab、Protein A/G Magnetic beads三者结合物的反应性。结果显示,B318L p Ab可以检测到上述细胞中内源过表达和外源表达的B318L蛋白以及结合在beads上的B318L蛋白,均出现约35 ku的特异性条带。上述结果表明,B318L蛋白是ASFV晚期表达的蛋白,且制备并纯化的B318L p Ab反应原性较强,既可以与HEK293T细胞中过表达的B318L蛋白也可以与ASFV感染的PAM中内源表达的B318L蛋白反应,反应原性较强,且可以用于Co-IP试验检测细胞中表达的B318L蛋白,本研究为深入研究ASFV B318L的生物学功能奠定了基础。

B318L,encoded by African swine fever virus(ASFV),is a late protein with Geranylgeranyl pyrophosphate synthase activity.In order to prepare a polyclonal antibody against B318L,this study amplified the ASFV B318L gene from the recombinant plasmid pCAGGS-HA-B318L by PCR and cloned into pGEX-6p-1 constructed a recombinant plasmid pGEX-6p-1-B318L.After PCR and sequencing identification,the recombinant plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.The recombinant protein was purified by GST affinity chromatography column,and the expression of the protein was detected by SDS-PAGE.The purification efficiency and reactivity of the recombinant protein were identified by western blot,And the protein concentration was measured by an ultraviolet spectrophotometer.The results showed that the specific bands appearing at approximately 60ku and the recombinant B318L protein(rB318L)mainly expressed in soluble form.After purification,a single specific band appeared at approximately 60ku,and the protein concentration was determined to be 10mg/mL by ultraviolet spectrophotometry.Mice were immunized with purified rB318L four times and the blood was collected one week after the third immunizations.The polyclonal antibody(pAb)was identified by western blot,and purified by Protein G affinity chromatography medium.The titer of pAb was detected by indirect ELISA.The purified antibody was identified by SDS-PAGE.The results showed that murine serum appeared specific bands at 60ku,and It reacted with ther B318L expressed by prokaryotic.The pAb contains both heavy and light chains with good purity,and the titer of pAb could reach 1∶32000.Different doses of pCAGGS-HA-B318L(1μg,2μg,4μg)were transfected into HEK293T cells,respectively,and the cells were collected at 24 hours later.Porcine alveolar macrophages(PAM)were infected with ASFV HLJ/18 strain(MOI 1)and the cells were collected at 6,12 and 24 hours after infection.Western blot and indirect immunofluorescence assay(IFA)(transfected with 1μg pCAGGS-HA-B318L)were used to detect the reactivity of prepared pAb.Western blot results showed that a specific band of about 35ku appeared in HEK293T cells,and the expression of B318L protein increased with the increase of transfection plasmid concentration.PAM infected with ASFV for 24 hours showed a specific band at 35ku,and no such band were observed at other time points.IFA results showed that red fluorescence appeared in HEK293T cells overexpressing B318L protein,PAM showed red fluorescence at 12 hours after infection with ASFV,and the amount of red fluorescence in PAM increased at 24 hours after infection.The prepared anti-B318L polyclonal antibody was used in immunoprecipitation(Co-IP)assay to detect the overexpression of B318L in 293T cells,the endogenous expression of B318L in PAM infection by ASFV,and the reactivity of the combination of the three of B318L protein expressed in the two cells and pAb and Protein A/G Magnetic beads with B318L pAb.Co-IP results showed that the anti-B318L pAb could detect B318L protein in the cells described above as well as B318L bound with beads,with a specific band of about 35ku.The results above indicated that B318L protein is a late expressed protein of ASFV,and the prepared and purified B318L pAb has strong reactivity.It could react with both overexpressed B318L in HEK293T cells and endogenous B318L protein expressed in PAM infected with ASFV,with strong reactivity.Moreover,it could be used for Co-IP assay to detect exogenous expression and endogenous B318L protein,laying a foundation for in-depth research of the biological function of ASFV B318L.