论文部分内容阅读
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 prokaryotic expression vector VP6 of rotavirus (RV) and prepare its polyclonal antibody. Methods A pair of specific primers was designed according to the sequence of VP6 gene of cattle RV UK strain (GenBank accession number: X53667.1). The VP6 full-length coding region of cattle RV UK strain was amplified by RT-PCR and cloned into vector p ET-28a. The recombinant plasmid p ET-28a-Bo-RV-VP6 was constructed and transfected into E.coli Rosetta (DE3). The recombinant protein was induced by IPTG. The recombinant protein was analyzed by SDS-PAGE and Western blot. The recombinant protein was mixed with Freund’s adjuvant and immunized guinea pigs. The immunized guinea pigs were immunized three times for 2 weeks at the interval of 2 weeks after the last immunization. Blood was collected from the heart and the serum was separated to prepare polyclonal antibody against VP6. Reactivity of RV. Results The recombinant plasmid pET-28a-Bo-RV-VP6 was identified by double enzyme digestion and sequencing. The recombinant proteins were expressed as inclusion bodies. The expressed proteins were 43,000, 34,000, 27,000 and 15,000 respectively. Four kinds of recombinant proteins with different relative molecular weights were obtained. The purity of purified recombinant proteins was over 95% Specific reaction. The prepared VP6 protein polyclonal antibody titer> 1: 7000, and can be identified RV natural VP6 protein. Conclusion Prokaryotic expression of bovine RV VP6 protein and the preparation of anti-VP6 polyclonal antibody provide the basis for the subsequent evaluation of RV vaccine efficacy and RV invasion mechanism.