H7N9流感病毒血凝素在昆虫杆状病毒表达系统中的表达及抗血清的制备

Expression of H7N9 influenza virus hemagglutinin in baculovirus expression system and preparation of the antiserum

目的:利用昆虫杆状病毒表达系统表达H7N9流感病毒血凝素(HA),以获得的重组血凝素(rHA1)蛋白免疫家兔制备抗rHA1血清,以用于单向免疫扩散(SRID)法检测H7N9流感病毒裂解疫苗HA含量。方法:构建含H7N9 HA1基因的重组杆状病毒,转染Sf9细胞表达rHA1蛋白。用rHA1蛋白免疫家兔获得抗rHA1血清。通过血凝抑制(HI)法和SRID法检测抗血清的HI效价、特异性和线性范围。结果:rHA1蛋白在昆虫杆状病毒表达系统中成功实现分泌表达,经纯化后纯度≥90%。不同剂量的rHA1蛋白免疫家兔后获得抗rHA1血清,HI效价分别为1 280和5 120。SRID试验中可与H7N9抗原国际参考品反应并产生沉淀环,10μg·m L^(-1)到40μg·m L^(-1)抗原浓度范围内沉淀环直径与抗原浓度线性正相关,R^2大于0.99,与H1N1、H3N2和B型流感病毒无交叉反应。用该免疫血清和H7N9抗血清国际参考品分别检测H7N9流感病毒裂解疫苗HA含量,两者无显著性差异。结论:在大流感发生时,昆虫杆状病毒表达系统表达的rHA1蛋白可以用于制备SRID试验用免疫血清,以用于H7N9流感病毒裂解疫苗HA含量测定。

Objective：To express H7N9 influenza virus hemagglutinin（HA）in a baculovirus expression system,so as to prepare anti-rHA1 serum by immunizing rabbits with the recombinant hemagglutinin（rHA1）protein,aiming to make HA content determination of H7N9 influenza vaccine（split virion）,by using single radial immunodiffusion（SRID）assay.Methods：The recombinant baculovirus containing H7N9 HA1 gene was constructed and transfected into Sf9 cell for rHA1 protein expression.Purifed rHA1 protein was used to prepare anti-rHA1 serum by immunizing the rabbits.（HI）titer,specificity and linear range of the antiserum were detected by hemagglutination inhibition（HI）method and SRID assay.Results：rHA1 protein was successfullyexpressed in the baculovirus expression system and purified with a purity above 90%.The anti-rHA1 sera were obtained after the rabbits were immunized with different dose of rHA1 protein.The HI titers were 5 120 and 1 280,respecitively.In the SRID assay,anti-rHA1 serum generated precipitation rings with NIBSC H7N9 reference antigen in a dose-related manner from 10 to 40 μg·mL-1 antigen concentration,with correlation coefficient values（R2）more than 0.99.Anti-rHA1 serum had no cross-reaction with H1N1,H3N2 and type B influenza virus.There was no significant difference between the HA concentrations of H7N9 influenza vaccine（split virion,inactivated）measured in SRID using anti-rHA1 serum and NIBSC H7N9 reference antiserum.Conclusion：When a pandemic occurs,the antiserum for SRID assay could be generated by immunizing rabbits with rHA1 protein expressed in baculovirus expression system,which can be used for HA content determination of H7N9 influenza vaccine（split virion,inactivated）.