摘要
【目的】构建瑶山亚种树鼩IFN-β和IFN-γ基因原核表达载体,并制备IFN-β和IFN-γ多克隆抗体,为进一步研究树鼩病毒感染动物模型打下基础。【方法】以瑶山亚种树鼩外周淋巴细胞为原材料,提取总RNA后经RT-PCR扩增瑶山亚种树鼩IFN-β和IFN-γ基因,亚克隆至质粒pcDNA3.1(+)上构建真核表达载体,并瞬时转染仓鼠肾细胞(BHK 21);同时亚克隆到原核载体pET-28a(+)上构建原核表达载体,转化大肠杆菌BL21(DE3)感受态细胞后以IPTG诱导表达融合蛋白IFN-β和IFN-γ。融合蛋白纯化后免疫小鼠制备多克隆抗体,并利用Western blotting和间接免疫荧光(IFA)检测分析其反应性。【结果】扩增获得的瑶山亚种树鼩IFN-β和IFN-γ基因片段分别为564和501 bp,亚克隆至质粒pcDNA3.1(+)上成功构建了真核表达载体pcDNA3.1-IFN-β和pcDNA3.1-IFN-γ,亚克隆到原核载体pET-28a(+)上成功构建获得原核表达载体pET-28a-IFN-β和pET-28a-IFN-γ。原核表达载体pET-28a-IFN-β和pET-28a-IFN-γ转化BL21(DE3)感受态细胞后在30℃下以0.5 mmol/L IPTG诱导6 h即获得融合蛋白IFN-β和IFN-γ,且融合蛋白主要以包涵体形式表达,以含2 mol/L尿素的洗涤液洗涤后可获得纯度相对较高的融合蛋白,且损失较少。纯化的融合蛋白IFN-β和IFN-γ经乳化后免疫小鼠,成功制备获得IFN-β和IFN-γ多克隆抗体,Western blotting和IFA检测结果均显示这2个多克隆抗体具有良好的反应性。【结论】通过原核表达系统可在大肠杆菌中成功诱导表达获得瑶山亚种树鼩IFN-β和IFN-γ,复性后的融合蛋白具有良好的反应性和免疫原性,免疫小鼠制备获得的多克隆抗体反应性良好且抗体效价高,可作为检测工具用于研究瑶山亚种树鼩病毒感染模型。
【Objective】To construct prokaryotic expression vector of IFN-βand IFN-γgenes in Tupaia belangeri yaoshanensis and prepare their polyclonal antibody,so as to lay the foundation for further research on animal models for viral infections of tree shrew.【Method】Peripheral lymphocytes of T.belangeri yaoshanensis were taken as material and to-tal RNA was extracted from them.IFN-βand IFN-γgenes,amplified by reverse transcription-polymerase chain reaction(RT-PCR),were subcloned into recombinant eukaryotic expression plasmids of pcDNA3.1(+)to censtruct eukaryotic ex-pression vector,and transiently transfected into renal cells of Cricetidae(BHK-21).At the same time,the genes were also were subcloned into prokaryotic vector pET-28a(+)to construct prokaryotic expression vector and transformed into Escherichia coli BL21(DE3)competent cell,and fusion protein of IFN-βand IFN-γwas induced by IPTG.Mice were immunized with purified recombinant proteins to obtain polyclonal antibodies of IFNβand IFNγ,and their reactivity were detected by Western blotting and indirect immunofluorescence assay(IFA).【Result】Fragments of IFN-βand IFN-γgenes in T.belangeri yaoshanensis obtained through amplification were 564 and 501 bp.They were subcloned to plasmids of pcDNA3.1(+),so eukaryotic expression vectors pcDNA3.1-IFN-βand pcDNA3.1-IFN-γwere constructed;and they were subcloned to prokaryotic vector of pET-28a(+),so prokaryotic expression vectors pET-28a-IFN-βand pET-28a-IFN-γwere constructed.After prokaryotic expression vectors pET-28a-IFN-βand pET-28a-IFN-γtransformed into BL21(DE3)competent cell,fusion proteins of IFNβand IFNγwere obtained induced by 0.5 mmol/L IPTG for 6 h at 30℃.The fusion proteins were expressed in a form of inclusion body.After washing with a washing solution containing 2 mol/L urea,fusion proteins of relatively high purity could be obtained with less loss.Mice were immunized with purified and emulsi-fied fusion proteins of IFNβand IFNγto obtain polyclonal antibodies of IFNβand IFNγ.Western blotting and IFA results showed that these two polyclonal antibodies were of good reactivity.【Conclusion】With prokaryotic expression system induced in E.coli,IFN-βand IFN-γin T.belangeri yaoshanensis was obtained;renatured fusion proteins are of good reactivity and immunogenicity.Polyclonal antibodies prepared through mice immunization were of good reactivity and high titer,and it can be taken as test tools for studying viral infection model of T.belangeri yaoshanensis.
作者
曹颖颖
李宝莹
王婷
梁亮
运晨霞
唐海波
冷静
CAO Ying-ying;LI Bao-ying;WANG Ting;LIANG Liang;YUN Chen-xia;TANG Hai-bo;LENG Jing(Guangxi University of Chinese Medicine/Guangxi Key Laboratory of Translational Medicine for Treating High incidence Infectious Diseases with Integrative Medicine/Guangxi Key Laboratory for Complementary and Alternative Medicine Experimental Animal Models,Nanning,Guangxi 530200,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2022年第6期1713-1723,共11页
Journal of Southern Agriculture
基金
国家自然科学基金项目(81560556)
广西自然科学基金项目(2020GXNSFAA297188)
广西高校千名中青年骨干教师培育计划项目(桂教师范〔2019〕81号)
广西中医药大学重点科研项目(2020ZD004)。
