FAdV-4 NP株F1蛋白原核表达及多克隆抗体的制备  

Prokaryotic expression of FAdV-4 F1 protein and preparation of anti-F1 polyclonal antibody

作　　者：陈媛[1] 屈贵蜀 彭尧舜 许丽惠[1] 王全溪[1] CHEN Yuan;QU Guishu;PENG Yaoshun;XU Lihui;WANG Quanxi(College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学动物科学学院

出　　处：《福建农林大学学报（自然科学版）》2019年第5期614-618,共5页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省自然科学基金(2019J01060639);福建农林大学科技发展基金(CXZX201789)

摘　　要：根据FAdV-4 NP株F1蛋白编码框的主要抗原位点,设计一对特异性引物,经PCR扩增、测序,分析表达效率后,进行密码子优化,以合成的新序列作为模板,构建重组表达质粒pET-32a-F1,将质粒转化到BL21中,筛选诱导条件,Western blot鉴定表达产物,将待纯化蛋白依次经过不同浓度的咪唑洗脱,纯化重组蛋白,最后将纯化后的蛋白免疫新西兰黄兔,收集血清并用间接ELISA法测定抗体效价.结果表明,所设计的特异性引物扩增了一条约699 bp的片段.PCR验证和酶切鉴定表明,pET-32a-F1被成功构建,并能够高效表达.重组质粒pET-32a-F1诱导的最佳IPTG浓度为0.3 mmol·??L^-1,最佳诱导时间为5 h,最佳诱导温度为37℃,咪唑洗脱浓度为80 mmol·??L^-1时,能获得较单一的洗脱蛋白.此外,本试验制备的F1多克隆抗体的效价可达1∶1 024 000.According to the main antigenic site of F1 protein coding frame of FAdV-4 strain NP,a pair of specific primers were de-signed for PCR amplification.Upon sequencing and analysis of the expression efficiency,the codons were optimized and applied to synthesize new sequence,which was set as PCR template.Then the recombinant expression plasmid pET-32a-F1 was constructed and transformed into BL21 competent cells to screen the induction conditions.The expression products were identified by Western blot.The recombinant protein was purified by affinity chromatography column method with different concentrations of imidazole.Fi-nally,the purified protein was used to immune New Zealand yellow rabbit,with the titer of the serum evaluated by indirect ELISA method.The results indicated that the designed specific primers amplified a fragment of about 699 bp.PCR identification and restric-tion enzyme digestion showed that pET-32a-F1 was successfully constructed and efficiently expressed.The optimal induction condi-tion was to induce under 37℃for 5 h with 0.3 mmol·??L^-1 sopropylβ-D-thiogalactoside.A single eluted protein could be obtained with 80 mmol·??L^-1 imidazole.The titer of anti-F1 polyclonal antibody reached 1∶1 024 000.

关 键 词：禽腺病毒血清4 型 F1 基因 原核表达 密码子优化 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]