| 摘要: |
| 【目的】克隆猪Jiv蛋白核心区的2 112 bp的基因片段，构建其原核表达载体后进行蛋白的表达和纯化，制备抗猪Jiv蛋白的多克隆抗体。【方法】利用RT-PCR方法，从猪脾脏组织RNA中扩增得到猪Jiv基因，将其克隆到pMD19-T载体并测序，以此为基础构建原核表达载体pET32a-Jiv，转化E.coli Rosetta(DE3)感受态细胞中，利用IPTG诱导表达，获得大量包涵体形式的猪Jiv融合蛋白。通过镍离子螯合层析柱对表达的目的蛋白进行纯化，对纯化后的包涵体蛋白进行透析复性，以每只新西兰白兔400 μg蛋白的初次免疫剂量和500 μg蛋白的加强免疫剂量进行免疫，共免疫5次，采集血清，采用间接ELISA法检测抗体效价达到一定水平后，收集抗体，利用Western blot检测抗体特异性。【结果】克隆得到长度为2 112 bp的猪Jiv基因片段；构建的pET32a-Jiv在E.coli Rosetta(DE3)感受态细胞中经诱导后高效表达，SDS-PAGE结果显示，纯化得到分子质量约为95 ku的猪Jiv融合蛋白；测得的猪Jiv蛋白多克隆抗体效价达1∶6 400；Western blot检测结果证实，所获得抗体能够有效地应用于猪Jiv蛋白抗原的检测。【结论】成功地克隆了猪Jiv基因，制备了抗猪Jiv蛋白的多克隆抗体。 |
| 关键词: 猪Jiv蛋白 原核表达 多克隆抗体 猪瘟病毒 |
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| 基金项目:西北农林科技大学基本科研业务费项目(QN2011109)； 国家自然科学基金项目(31172339) |
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| Cloning and expression of swine Jiv gene and preparation of its polyclonal antibody |
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IU Wei,GUO Kang-kang,LIN Zhi,et al
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| Abstract: |
| 【Objective】This study aimed to clone the 2 112 bp Jiv gene from core area of swine Jiv protein,construct the prokaryotic expression vector for the expression and purification of fusion protein,and prepare polyclonal antibody against swine Jiv protein.【Method】The Jiv gene was amplified by RT-PCR from swine spleen tissues and cloned into pMD19-T vector to sequence.It also acted as template to construct prokaryotic expression vector pET32a-Jiv and the recombinant vector was transformed into E.coli Rosetta (DE3).Swine Jiv fusion proteins with the inclusion form were obtained inducing by IPTG and were purified by Ni2+ charged column.After purification,inclusion body was refolded through dialysis renaturation.400 μg protein was injected into each New Zealand white rabbit as the first immunization and 500 μg protein as strengthening immunization was used for a total of five times.When titers of antibodies detected by indirect ELISA reached a certain level,the antibodies were collected and identified by Western blot.【Result】The fragment of swine Jiv gene with the length of 2 112 bp was successfully obtained.The constructed pET32a-Jiv was highly expressed in E.coli Rosetta (DE3) after induction by IPTG.SDS-PAGE showed that the purified fusion protein was about 95 ku.The titer of antibodies was 1∶6 400 and Western blot showed that the obtained antibodies could be used to detect swine Jiv protein effectively.【Conclusion】Swine Jiv gene was cloned and the polyclonal antibodies against swine Jiv protein were prepared. |
| Key words: swine Jiv protein prokaryotic expression polyclonal antibody classical swine fever virus |
