1型鸭甲肝病毒VP3蛋白的抗血清中和活性分析及B细胞表位鉴定

Neutralizing Activity Analysis of VP3 Antiserums and B-cell Epitopes Identification of VP3 Protein form Duck Hepatitis A Virus Type 1

旨在探究1型鸭甲肝病毒(DHAV-1)VP3蛋白抗血清的中和活性并鉴定VP3的线性B细胞表位。利用pGEX-4T-1表达载体,在BL21(DE3)宿主菌中原核表达DHAV-1 VP3基因,以切胶纯化出的蛋白质为抗原免疫兔制备多克隆抗体,通过鸡胚中和试验对多抗的中和效价进行检测;采用Karplus&Schulz、Emini、Jameson-Wolf和Parker方法分别对柔韧性、表面可及性、抗原性及亲水性进行分析,得到了4条候选线性B细胞表位,以制备的兔抗VP3多克隆抗体为一抗,通过间接ELISA方法对人工合成的B细胞表位进行鉴定,并进一步用临床鸭血清样品对鉴定的B细胞表位的抗体检测能力进行评估。结果显示,VP3在BL21(DE3)中以包涵体形式表达,大小约54ku,Western blot分析表明重组蛋白质具有较好的反应原性。制备的兔抗VP3多克隆抗体的琼扩效价达到1∶16,并能中和DHAV-1,中和效价为1∶39;利用间接ELISA鉴定出GKRKPCRRPIHKPKNPPQEP(1—20aa)、FNTGRYQMSWYPIADGEQSL(131—150aa)和VNSSAPSNID(200—209aa)为VP3的B细胞表位,抗体检测能力试验结果显示表位肽可检测临床DHAV-1鸭血清。本研究表明DHAV-1VP3的抗血清具备一定的中和活性,1—20aa、131—150aa和200—209aa为VP3的B细胞表位且具有临床应用前景。

This study aimed at researching neutralizing activity of VP3 antiserums and determining B-cell epitopes of VP3 protein from duck hepatitis A virus type 1（DHAV-1）.The pGEX-4T-1expression plasmid was used to express VP3 gene of DHAV-1in E.coli BL21（DE3）.The expressed recombinant VP3 protein was purified by gel extraction and was used as immunogen to rabbit to prepare polyclonal antibodies.Chicken embryo neutralization test was conducted to detect the neu-tralizing titer of polyclonal antibodies.Moreover,KarplusSchulz,Emini,Jameson-Wolf and Parker method were used to analyze the flexibility,surface accessibility,antigenicity and hydrophilicity of the VP3 protein,respectively.This contributed to obtain four probable B-cell epitopes for VP3 protein.The rabbit anti-VP3 polyclonal antibody was used as the first antibody of indirect ELISA to identity the synthetic peptides.Furthermore,apanel of clinical duck serum samples was used to evaluate capacity of the identified B-cell epitopes for antibodies detection.The results showed that the recombinant VP3 protein was expressed as inclusion body in E.coli BL21（DE3）with a molecular weight about 54 kD,and proved to be with good reactogenicity by Western blot analysis.The prepared polyclonal antibodies reached an titer of 1∶16by AGP test,it could neutralize DHAV-1and reached a titer of 1∶39.Furthermore,the B-cell epitopes identified by indirect ELISA were GKRKPCRRPIHKPKNPPQEP（1-20aa）,FNTGRYQMSWYPIADGEQSL（131-150aa）and VNSSAPSNID（200-209aa）.The test for antibody detection ability revealed the epitopes were capable of detecting clinical duck antiserums of DHAV-1.This study proved antiserums to VP3 protein of DHAV-1with neutralizing activity and identified VP3＇s three promising B-cell epitopes,1-20 aa,131-150 aa and 200-209 aa,for clinical use.