IBV S1蛋白的原核表达及其单克隆抗体的制备

采用RT-PCR方法,以IBVM41毒株的S1基因为模板扩增出了大小为468bp的目的片段(命名为poly156S1),该片断保守性、抗原性较好。将获得的目的基因定向克隆到表达载体pET-32a-c(+)中,获得了重组质粒pET-32a-c(+)-poly156S1。将该重组质粒转入表达菌Origami(DE3)中,用IPTG进行诱导表达,获得了大小约为34KD的重组蛋白poly156S1。Western-blot证实该重组蛋白能与IBV阳性鸡血清发生特异性反应。用纯化好的重组蛋白poly156S1免疫BALB/c小鼠,按常规方法进行单克隆抗体的制备,成功获得了针对M41S1蛋白的3株单克隆杂交瘤细胞8D2、8D6、10F9,其IgG亚类分别为IgG1、IgG2a、IgG2b,轻链均为κ型。按常规方法对各株单克隆杂交瘤细胞进行了小鼠腹水单抗的制备与纯化,并用以重组蛋白poly156S1为包被抗原的间接ELISA对纯化后的腹水单抗进行了效价测定,及其与重组蛋白poly156S1亲和力的分析。结果表明:腹水单抗8D2、8D6、10F9的ELISA效价分别达到1.78×218,1.37×221,1.31×216;单抗8D6与重组蛋白poly156S1的亲和力最高。Western-blot进一步证实单抗8D6与重组蛋白poly156S1具有良好的反应性。

IBV S1 potein Prokaryotic Expression and its Monoclonal Antibody Preparation

Using RT-polymerase chain reaction (PCR) to IBV M41 strain of S1 gene for template amplification out of the size for 468 bp purpose fragment (named poly156 S1),this fragment conservative,antigenicity better.Will get purpose gene orientation cloning to expression vector pET-32 a c (+),won the recombinant plasmid pET-32 a-c (+) poly156 S1.The recombinant plasmid into express bacterium Origami (DE3),use IPTG induction expression,won the size of 34 KD of recombinant protein poly156 S1.Western-blot confirmed that the recombinant protein with IBV positive chicken serum happen specific reaction.Purification of recombinant proteins with good poly156 S1 immune BALB/c mice,according to the conventional methods for the preparation of monoclonal antibodies,successful in M41 S1 protein reason monoclonal hybridoma 8 d2,8 d6,10 f9,its IgG subgenera were IgG1,IgG2a,IgG2b,light chain are κ type.According to the conventional method of each plant monoclonal antibody hybridoma cells mice ascites single preparation and purification of resistance,and used to recombinant protein poly156 S1 as coating antigen of indirect ELISA for purification of ascites after the single resistance potency determination,and the analysis of the recombinant protein poly156 S1 affinity.The results show that ascites single resistance to 8 d2,8 d6,10 f9 of ELISA valence reached 1.78×2 18,1.37×2 21,1.31×2 16 ;Single resistance to 8 d6 and recombinant protein poly156 S1 highest affinity.Western-blot further confirmed that single resistance to 8 d6 and recombinant protein poly156 S1 has good reactivity.