弓形虫SAG5B和SAG1基因的克隆表达及其对小鼠免疫保护性研究

发布时间：2018-01-21 12:43

  本文关键词： 弓形虫 疫苗 克隆 表达 免疫保护性　出处：《安徽医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:克隆刚地弓形虫RH株表膜的sag1以及一种新发现的基因sag5b,并构建原核表达载体pET28a/sag1和pET28a/sag5b ,表达弓形虫表膜蛋白SAG1和SAG5B。利用此重组蛋白免疫小鼠,观察对小鼠的抗弓形虫感染的能力。同时对照比较RH株弓形虫基因组与Praugniud株弓形虫基因组中sag5b基因,用于虫株毒力鉴别的可能性。方法:复苏本室液氮保种的RH株弓形虫速殖子,腹腔接种Balb/c小鼠,转种3代,抽取腹水,收集、纯化弓形虫速殖子,并用蛋白酶K裂解法提取基因组DNA,同时制备速殖子粗抗原并免疫新西兰白兔,收集兔抗弓形虫多抗血清。剖杀脑组织中含有Praugniud株弓形虫包囊的小鼠,获取含有包囊的脑组织。引物设计时分别在引物两端引入EcoRI和XhoI以及EcoRI和HindIII酶切位点。PCR扩增出弓形虫sag5b以及sag1基因片断,目的基因sag5b以及sag1基因片断插入克隆载体pGEM-T,提取重组质粒,双酶切鉴定并获得目的基因,插入原核表达载体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,SDS-PAGE和Western blotting验证表达量和免疫活性,并用Lowry法测定纯化rSAG5B及rSAG1蛋白浓度。将两种纯化的蛋白以及弓形虫粗抗原分别皮下免疫Balb/c小鼠,再进行攻击感染,以观察小鼠的存活情况。以佐剂为空白对照。结果:利用PCR从RH株弓形虫基因组中克隆出1104bp的sag5b目的基因片段。成功地将其克隆入pET28a。sag5b-pET28a经EcoRI和XhoI双酶切,获得与目的基因大小相一致的基因片段,测序结果与GenBank比对sag5b同源性100%。含sag5b-pET28a的宿主菌E.coliBL21(DE3)经IPTG诱导后高效表达rSAG5B蛋白。分别以RH株弓形虫基因组以及含有Praugniud株弓形虫包囊的脑组织为模板,PCR法扩增sag5b,只有在RH株基因组中发现该基因。此外,利用PCR从RH株弓形虫基因组中克隆出1101bp的sag1目的基因片段。将其克隆入pET28a载体。sag1-pET28a经EcoRI和HindIII双酶切,获得与目的基因大小相一致的基因片段,测序结果与GenBank比对sag1同源性100%。含sag1-pET28a的宿主菌E.coliBL21(DE3)经IPTG诱导后高效表达rSAG1。上述两种重组蛋白经Ni2+亲和层析法纯化获得了高纯度的rSAG5B和rSAG1蛋白。SDS-PAGE检测其分子量:rSAG5B为43KDa, rSAG1为30KDa,二者分别与各自的预期分子量大小相符。Western blotting显示:rSAG5B与rSAG1蛋白都能够被抗弓形虫的多抗血清中的相应抗体识别,获得了两种纯化的具有特异免疫反应的重组蛋白。将这两个纯化的重组蛋白以及制备的弓形虫粗抗原分别与福氏佐剂乳化后皮下免疫Balb/c小鼠,同时以福氏佐剂为空白对照。每只小鼠的抗原用量为20μg,两周后加强一次,佐剂改为福氏不完全佐剂。末次免疫后一个月,用RH株弓形虫速殖子腹腔注射小鼠进行攻击感染,统计学比较发现rSAG5B和rSAG1与粗抗原对小鼠的免疫保护作用没有显著性差异(P0.05),而均比空白对照组(未免疫组)生存时间长,有显著性差异(P0.05)。结论:成功地从弓形虫RH株基因组中获取了sag5b基因以及sag1,构建了sag5b-pET28a/sag1-pET28a重组质粒,并获得了高效表达;对比RH株基因组与Praugniud株基因组表明,sag5b可能作为鉴别强毒株与弱毒株的遗传标志。制备RH株弓形虫速殖子粗抗原并免疫新西兰白兔获得抗弓形虫多抗血清。粗抗原与获得的纯化重组蛋白分别免疫Balb/c小鼠,并与空白做比较发现,rSAG5B以及rSAG1均对小鼠有免疫保护作用,且与粗抗原的作用相仿。对弓形虫新发现的表膜蛋白SAG5B对小鼠的免疫保护性的研究,有助于开发有效的预防弓形虫感染的疫苗。
[Abstract]:Objective: to clone RH strain of Toxoplasma gondii surface membrane SAG1 and a newly discovered gene sag5b, and construct a prokaryotic expression vector pET28a/sag1 and pET28a/sag5b, the expression of Toxoplasma surface membrane protein SAG1 and SAG5B. using the recombinant protein immunized mice were observed in mice against Toxoplasma gondii infection ability. At the same time compared with RH strain Toxoplasma gondii genome and Praugniud strains of Toxoplasma gondii sag5b gene in the genome, possibility for virulence identification. Methods: the recovery room liquid nitrogen for conservation of RH strains of Toxoplasma gondii tachyzoites were inoculated into Balb/c mice, 3 generation ascites collection, purification of Toxoplasma gondii tachyzoites and K protease cracking method to extract genomic DNA, and preparation of tachyzoite crude antigen and immune rabbit polyclonal antibodies against Toxoplasma gondii were collected. Killed in the brain tissue of mice with Toxoplasma cysts of the Praugniud strain, for containing cysts of the brain. The primers were designed respectively in the introduction of EcoRI and XhoI and the ends of primers EcoRI and HindIII restriction sites were amplified by.PCR and sag5b of Toxoplasma gondii SAG1 gene fragment, sag5b gene and SAG1 gene fragments into the cloning vector pGEM-T, recombinant plasmid, double enzyme digestion and target genes, inserted into prokaryotic expression vector pET28a, recombinant double enzyme digestion, PCR and sequencing, transformed into Escherichia coli E.coli (DE3) BL21 and IPTG expression. The recombinant protein was purified by affinity chromatography, and immune activity of SDS-PAGE and blotting verification Western expression, and the Lowry method for the determination of purified rSAG5B and rSAG1 protein concentration. Two kinds of purified protein and crude antigen of Toxoplasma gondii Balb/c mice were immunized subcutaneously respectively, and then attacked by infected mice. The adjuvant control group. Results: the use of PCR 1104bp cloned from RH strain of Toxoplasma gondii genome The sag5b gene fragment. Successfully cloned into pET28a.sag5b-pET28a by EcoRI and XhoI double enzyme digestion, and the size of the target gene fragment consistent with the sequencing results, compared with the GenBank sag5b homologous host bacteria E.coliBL21 100%. containing sag5b-pET28a (DE3) induced with IPTG high expression of rSAG5B protein with RH strain. Toxoplasma gondii genome and contains the brain tissue of Praugniud strains of Toxoplasma gondii cysts as template, amplified sag5b PCR, only to find the gene in the genome of RH. In addition, the use of PCR cloned SAG1 gene fragment of 1101bp from RH strain of Toxoplasma gondii genome. The gene was cloned into pET28a plasmid.Sag1-pET28a by EcoRI and HindIII double enzyme cut, with the size of the target gene fragment was consistent with the sequencing results, compared with the GenBank SAG1 homologous host bacteria E.coliBL21 100%. containing sag1-pET28a (DE3) induced by IPTG after high table RSAG1. of the two recombinant protein by Ni2+ affinity chromatography to obtain the high purity of rSAG5B and rSAG1 to detect the.SDS-PAGE protein and its molecular weight: rSAG5B 43KDa, rSAG1 30KDa, two respectively with the expected molecular weight of each with the size of.Western blotting showed that the corresponding antibody recognition rSAG5B and rSAG1 protein can be anti Toxoplasma polyclonal antibody in serum, obtained two kinds of purified recombinant protein had specific immune reaction. The two purified recombinant protein and preparation of Toxoplasma gondii crude antigen respectively with Freund's adjuvant emulsion after subcutaneous immunization of Balb/c mice. At the same time with Freund's adjuvant control group. Each mouse antigen was 20 g, after two weeks of time to strengthen, adjuvant incomplete Freund's adjuvant. One month after the last immunization with Toxoplasma gondii RH strain tachyzoites in mice after intraperitoneal injection of attack infection, statistical comparison showed that rSAG5B There is no significant difference between rSAG1 and crude antigen in mice with immune protective effect (P0.05), and compared with the blank control group (non immune group) survival time, there was a significant difference (P0.05). Conclusion: the success from the genome of RH strain Toxoplasma gondii obtained sag5b gene and SAG1, the construction of the recombinant sag5b-pET28a/sag1-pET28a the plasmid, and was highly expressed; comparative genome of RH and Praugniud genome showed that sag5b may be used as a genetic marker for identification of virulent and attenuated. Preparation of Toxoplasma gondii RH strain tachyzoites and crude antigen to immunize New Zealand rabbits obtained anti Toxoplasma antiserum. Crude antigen and purification of recombinant protein obtained respectively Balb/c mice, and compared with the blank, rSAG5B and rSAG1 have protective effect on mice, and the effect of crude antigen on immune protection. Similar to newly discovered Toxoplasma surface membrane protein SAG5B in rats The study of sex helps to develop effective vaccines to prevent Toxoplasma infection.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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