弓形虫次黄嘌呤—黄嘌呤—鸟嘌呤磷酸核糖转移酶（HXGPRT）和腺苷激酶（AK）基因的克

发布时间：2018-01-22 01:01

  本文关键词： 弓形虫 次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶 腺苷激酶 克隆 表达 免疫 多克隆抗体　出处：《安徽医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:克隆弓形虫RH株次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶(HXGPRT)和腺苷激酶(AK)基因,构建原核表达载体pET28a/HXGPRT和pET28a/AK,表达HXGPRT和AK重组蛋白,利用此重组蛋白免疫新西兰白兔制备多抗血清。方法:复苏本室液氮保种的弓形虫RH株速殖子,腹腔接种BALB/c小鼠,转种3代,抽取腹水,收集、纯化弓形虫株速殖子,提取总RNA,在设计合成的引物中引入BamHI和XhoI酶切位点。应用RT-PCR扩增弓形虫HXGPRT,AK基因片段,目的基因HXGPRT插入克隆载体pMD18-T,目的基因AK插入克隆载体pGEM-T,提取重组质粒,BamHI和XhoI双酶切获得目的基因,插入原核表达质粒pET28a中,重组子双酶切、PCR和测序鉴定,转化大肠杆菌E.coli BL21(DE3)并以IPTG诱导表达。亲和层析法纯化重组蛋白抗原,SDS-PAGE和Western blotting验证表达量和免疫活性,改良的Bradford(考马斯亮蓝)法测定纯化rHXGPRT和rAK蛋白浓度。免疫新西兰白,收集多抗血清,ELISA测定多抗滴度,Western blotting鉴定免疫活性。结果:RT-PCR从弓形虫RH株RNA中扩增出693bp的HXGPRT和1092bp的AK目标基因片段。成功地将其克隆入pET28a。pET28a/HXGPRT和pET28a/AK经BamHI和XhoI双酶切,获得与目标基因大小相一致的基因片段,测序结果与GenBank比对HXGPRT同源性100%,AK同源性99.9%。含pET28a/HXGPRT和pET28a/AK的宿主菌E.coliBL21(DE3)经IPTG诱导后高效表达上述2个重组蛋白,经Ni~(2+)亲和层析法纯化获得了高纯度的rHXGPRT和rAK蛋白。SDS-PAGE检测其分子量:rHXGPRT为26.4kDa,rAK为38.357kDa,二者与各自预期分子量大小相符。Western blotting显示:rHXGPRT能够被弓形虫患者血清中的IgG抗体和Torch-ELISA试剂合中的Tox-IgG阳性控制血清识别,rAK能够被Torch试剂合中的Tox-IgG阳性控制血清识别,获得了两种纯化的具有特异免疫反应性的重组蛋白。rHXGPRT和rAK分别免疫新西兰白兔,获得ELISA滴度为1:10~7和1:10~6。两种多价抗血清,Westernblotting显示,每种多价血清不仅能和其对应的重组蛋白而且和虫体内相应蛋白发生特异性免疫反应。结论:成功地从弓形虫RH株基因组中获取了HXGPRT和AK基因,构建了pET28a/HXGPRT和pET28a/AK重组质粒,并获得了高效表达;重组蛋白免疫新西兰白兔获得了两种高效价多克隆抗体。本研究为对弓形虫嘌呤核酸代谢及基因沉默的研究,以及研发标准化弓形虫免疫血清诊断试剂打下了基础。
[Abstract]:Objective: to clone HXGPRT and AK gene of RH strain of Toxoplasma gondii. The prokaryotic expression vectors pET28a/HXGPRT and pET28a / AK were constructed to express HXGPRT and AK recombinant proteins. The recombinant protein was used to immunize New Zealand white rabbits to prepare polyantiserum. Methods: Toxoplasma gondii RH strain Toxoplasma gondii Toxoplasma were resuscitated and intraperitoneally inoculated into BALB/c mice for 3 generations. Ascites were extracted and collected. Toxoplasma gondii Tachyzoites were purified, total RNAs were extracted, and BamHI and XhoI restriction sites were introduced into the primers designed and synthesized. RT-PCR was used to amplify the HXGPRTT-AK gene fragment of Toxoplasma gondii. Objective HXGPRT was inserted into pMD18-T and AK was inserted into cloning vector pGEM-T. the recombinant plasmid was digested with BamHI and XhoI to obtain the target gene. The recombinant plasmid was inserted into the prokaryotic expression plasmid pET28a and identified by double enzyme digestion and sequencing. E. coli BL21 (DE3) was transformed into E. coli BL21 (DE3) and expressed by IPTG. The recombinant protein antigen was purified by affinity chromatography. SDS-PAGE and Western blotting were used to verify the expression and immune activity. The purified rHXGPRT and rAK protein concentrations were determined by modified Bradford method. New Zealand white was immunized, and polyclonal antibodies were collected to determine the titer of polyclonal antibodies by enzyme-linked immunosorbent assay (Elisa). Western blotting was used to identify the immune activity. RT-PCR amplified 693bp HXGPRT and 1092bp AK target gene from RNA of Toxoplasma gondii RH strain, and cloned them into pET28a.pET28a successfully. / HXGPRT and pET28a/AK were digested by BamHI and XhoI. The gene fragment which was consistent with the size of the target gene was obtained and sequenced. The HXGPRT homology was 100% compared with GenBank. AK homology 99.9. E. coli BL21DE3 containing pET28a/HXGPRT and pET28a/AK. The two recombinant proteins were highly expressed after induction by IPTG. High purity rHXGPRT and rAK protein were purified by Ni~(2 affinity chromatography. SDS-PAGE showed that the molecular weight of rHXGPRT and rAK protein was 26.4kDa. RAK was 38.357 kDa. Western blotting showed that the molecular weight of the two molecules was in accordance with their expected molecular weight. RHXGPRT can be recognized by the IgG antibody in the serum of Toxoplasma gondii patients and the Tox-IgG positive control serum in the Torch-ELISA reagent combination. RAK can be recognized by Tox-IgG positive control serum in Torch reagent combination. Two purified recombinant proteins, rHXGPRT and rAK, were obtained to immunize New Zealand white rabbits. The titers of ELISA were 1: 10 ~ 7 and 1: 10 ~ (6) respectively. Two kinds of multivalent antiserum were detected by Western blotting. Each polyvalent serum can react with the corresponding recombinant protein and the corresponding protein. Conclusion: HXGPRT and AK genes were successfully obtained from the genome of Toxoplasma gondii RH strain. The recombinant plasmids of pET28a/HXGPRT and pET28a/AK were constructed and highly expressed. Two high titers polyclonal antibodies were obtained from New Zealand white rabbits immunized with recombinant proteins. This study was designed to study the metabolism and gene silencing of Toxoplasma gondii purine nucleic acid. And the research and development of standardized Toxoplasma gondii immune serum diagnostic reagent laid the foundation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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