發(fā)布時(shí)間：2018-01-22 01:01

  本文關(guān)鍵詞： 弓形蟲 次黃嘌呤-黃嘌呤-鳥嘌呤磷酸核糖轉(zhuǎn)移酶 腺苷激酶 克隆 表達(dá) 免疫 多克隆抗體　出處：《安徽醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:克隆弓形蟲RH株次黃嘌呤-黃嘌呤-鳥嘌呤磷酸核糖轉(zhuǎn)移酶(HXGPRT)和腺苷激酶(AK)基因,構(gòu)建原核表達(dá)載體pET28a/HXGPRT和pET28a/AK,表達(dá)HXGPRT和AK重組蛋白,利用此重組蛋白免疫新西蘭白兔制備多抗血清。方法:復(fù)蘇本室液氮保種的弓形蟲RH株速殖子,腹腔接種BALB/c小鼠,轉(zhuǎn)種3代,抽取腹水,收集、純化弓形蟲株速殖子,提取總RNA,在設(shè)計(jì)合成的引物中引入BamHI和XhoI酶切位點(diǎn)。應(yīng)用RT-PCR擴(kuò)增弓形蟲HXGPRT,AK基因片段,目的基因HXGPRT插入克隆載體pMD18-T,目的基因AK插入克隆載體pGEM-T,提取重組質(zhì)粒,BamHI和XhoI雙酶切獲得目的基因,插入原核表達(dá)質(zhì)粒pET28a中,重組子雙酶切、PCR和測序鑒定,轉(zhuǎn)化大腸桿菌E.coli BL21(DE3)并以IPTG誘導(dǎo)表達(dá)。親和層析法純化重組蛋白抗原,SDS-PAGE和Western blotting驗(yàn)證表達(dá)量和免疫活性,改良的Bradford(考馬斯亮藍(lán))法測定純化rHXGPRT和rAK蛋白濃度。免疫新西蘭白,收集多抗血清,ELISA測定多抗滴度,Western blotting鑒定免疫活性。結(jié)果:RT-PCR從弓形蟲RH株RNA中擴(kuò)增出693bp的HXGPRT和1092bp的AK目標(biāo)基因片段。成功地將其克隆入pET28a。pET28a/HXGPRT和pET28a/AK經(jīng)BamHI和XhoI雙酶切,獲得與目標(biāo)基因大小相一致的基因片段,測序結(jié)果與GenBank比對HXGPRT同源性100%,AK同源性99.9%。含pET28a/HXGPRT和pET28a/AK的宿主菌E.coliBL21(DE3)經(jīng)IPTG誘導(dǎo)后高效表達(dá)上述2個(gè)重組蛋白,經(jīng)Ni~(2+)親和層析法純化獲得了高純度的rHXGPRT和rAK蛋白。SDS-PAGE檢測其分子量:rHXGPRT為26.4kDa,rAK為38.357kDa,二者與各自預(yù)期分子量大小相符。Western blotting顯示:rHXGPRT能夠被弓形蟲患者血清中的IgG抗體和Torch-ELISA試劑合中的Tox-IgG陽性控制血清識別,rAK能夠被Torch試劑合中的Tox-IgG陽性控制血清識別,獲得了兩種純化的具有特異免疫反應(yīng)性的重組蛋白。rHXGPRT和rAK分別免疫新西蘭白兔,獲得ELISA滴度為1:10~7和1:10~6。兩種多價(jià)抗血清,Westernblotting顯示,每種多價(jià)血清不僅能和其對應(yīng)的重組蛋白而且和蟲體內(nèi)相應(yīng)蛋白發(fā)生特異性免疫反應(yīng)。結(jié)論:成功地從弓形蟲RH株基因組中獲取了HXGPRT和AK基因,構(gòu)建了pET28a/HXGPRT和pET28a/AK重組質(zhì)粒,并獲得了高效表達(dá);重組蛋白免疫新西蘭白兔獲得了兩種高效價(jià)多克隆抗體。本研究為對弓形蟲嘌呤核酸代謝及基因沉默的研究,以及研發(fā)標(biāo)準(zhǔn)化弓形蟲免疫血清診斷試劑打下了基礎(chǔ)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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