牛轮状病毒VP6蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of VP6 protein of bovine rotavirus and preparation of polyclonal antibody

目的原核表达牛轮状病毒(rotavirus,RV)VP6蛋白,并制备其多克隆抗体。方法根据Gen Bank中登录的牛RV UK株VP6基因序列(X53667.1)设计一对特异性引物,RT-PCR扩增牛RV UK株VP6全长编码区片段,克隆至载体p ET-28a,构建重组表达质粒p ET-28a-Bo-RV-VP6,转染至E.coli Rosetta(DE3),IPTG诱导表达,重组蛋白进行SDS-PAGE和Western blot分析。将重组蛋白与弗氏佐剂混合后免疫豚鼠,共免疫3次,每次间隔2周,末次免疫后2周,经心脏采血,分离血清,制备VP6多克隆抗体,Western blot法检测其与牛RV的反应原性。结果经双酶切和测序鉴定,重组表达质粒p ET-28a-Bo-RV-VP6构建正确。重组蛋白以包涵体的形式表达,表达产物分别为43 000、34 000、27 000和15 000 4种不同相对分子质量的重组蛋白,纯化后纯度可达95%以上,可与牛RV阳性血清发生特异性反应。制备的VP6蛋白多克隆抗体效价>1∶7 000,且可识别RV天然VP6蛋白。结论原核表达了牛RV VP6蛋白,并制备了抗VP6多克隆抗体,为后续RV疫苗效力评价及RV侵入机理等方面的研究奠定了基础。

Objective To express the VP6 protein of rotavirus（RV） in prokaryotic cells and prepare its polyclonal antibody.Methods Based on the sequence of bovine RV UK strain in Gen Bank（X53667.1）,a pair of specific primers were designed,with which the open reading fragment（ORF） of VP6 was amplified by RT-PCR and cloned into vector p ET-28 a.The constructed recombinant plasmid p ET-28a-Bo-RV-VP6 was transfected to E.coli Rosetta（DE3） and induced by IPTG.The expressed recombinant protein was analyzed by SDS-PAGE and Western blot.Guinea pigs were immunized the mixture of the recombinant protein and Freund adjuvant for 3 times each at an interval of 2 weeks,of which the serum samples were collected 2 weeks after the last immunization.The prepared polyclonal antibody against VP6 was tested for reactogenicity with bovine RV by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid p ET-28a-Bo-RV-VP6 was constructed correctly.Recombinant VP6 proteins,with relative molecular masses of 43 000,34 000,27 000 and 15 000 respectively,were expressed each in a form of inclusion body,and reached purities of more than 95% after purification,which showed specific reactions with RV positive bovine sera.The prepared polyclonal antibodies against VP6 protein reached a titer of more than 1 ∶ 7 000,and recognized the natural VP6 protein of RV.Conclusion The bovine RV VP6 protein was expressed by using prokaryotic expression system,and polyclonal antibody against VP6 was successfully prepared,which laid a foundation of further evaluation on efficacy of RV vaccine and research on the invasion mechanism of RV.