本文關(guān)鍵詞： 弓形蟲 疫苗 克隆 表達(dá) 免疫保護性　出處：《安徽醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:克隆剛地弓形蟲RH株表膜的sag1以及一種新發(fā)現(xiàn)的基因sag5b,并構(gòu)建原核表達(dá)載體pET28a/sag1和pET28a/sag5b ,表達(dá)弓形蟲表膜蛋白SAG1和SAG5B。利用此重組蛋白免疫小鼠,觀察對小鼠的抗弓形蟲感染的能力。同時對照比較RH株弓形蟲基因組與Praugniud株弓形蟲基因組中sag5b基因,用于蟲株毒力鑒別的可能性。方法:復(fù)蘇本室液氮保種的RH株弓形蟲速殖子,腹腔接種Balb/c小鼠,轉(zhuǎn)種3代,抽取腹水,收集、純化弓形蟲速殖子,并用蛋白酶K裂解法提取基因組DNA,同時制備速殖子粗抗原并免疫新西蘭白兔,收集兔抗弓形蟲多抗血清。剖殺腦組織中含有Praugniud株弓形蟲包囊的小鼠,獲取含有包囊的腦組織。引物設(shè)計時分別在引物兩端引入EcoRI和XhoI以及EcoRI和HindIII酶切位點。PCR擴增出弓形蟲sag5b以及sag1基因片斷,目的基因sag5b以及sag1基因片斷插入克隆載體pGEM-T,提取重組質(zhì)粒,雙酶切鑒定并獲得目的基因,插入原核表達(dá)載體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,SDS-PAGE和Western blotting驗證表達(dá)量和免疫活性,并用Lowry法測定純化rSAG5B及rSAG1蛋白濃度。將兩種純化的蛋白以及弓形蟲粗抗原分別皮下免疫Balb/c小鼠,再進行攻擊感染,以觀察小鼠的存活情況。以佐劑為空白對照。結(jié)果:利用PCR從RH株弓形蟲基因組中克隆出1104bp的sag5b目的基因片段。成功地將其克隆入pET28a。sag5b-pET28a經(jīng)EcoRI和XhoI雙酶切,獲得與目的基因大小相一致的基因片段,測序結(jié)果與GenBank比對sag5b同源性100%。含sag5b-pET28a的宿主菌E.coliBL21(DE3)經(jīng)IPTG誘導(dǎo)后高效表達(dá)rSAG5B蛋白。分別以RH株弓形蟲基因組以及含有Praugniud株弓形蟲包囊的腦組織為模板,PCR法擴增sag5b,只有在RH株基因組中發(fā)現(xiàn)該基因。此外,利用PCR從RH株弓形蟲基因組中克隆出1101bp的sag1目的基因片段。將其克隆入pET28a載體。sag1-pET28a經(jīng)EcoRI和HindIII雙酶切,獲得與目的基因大小相一致的基因片段,測序結(jié)果與GenBank比對sag1同源性100%。含sag1-pET28a的宿主菌E.coliBL21(DE3)經(jīng)IPTG誘導(dǎo)后高效表達(dá)rSAG1。上述兩種重組蛋白經(jīng)Ni2+親和層析法純化獲得了高純度的rSAG5B和rSAG1蛋白。SDS-PAGE檢測其分子量:rSAG5B為43KDa, rSAG1為30KDa,二者分別與各自的預(yù)期分子量大小相符。Western blotting顯示:rSAG5B與rSAG1蛋白都能夠被抗弓形蟲的多抗血清中的相應(yīng)抗體識別,獲得了兩種純化的具有特異免疫反應(yīng)的重組蛋白。將這兩個純化的重組蛋白以及制備的弓形蟲粗抗原分別與福氏佐劑乳化后皮下免疫Balb/c小鼠,同時以福氏佐劑為空白對照。每只小鼠的抗原用量為20μg,兩周后加強一次,佐劑改為福氏不完全佐劑。末次免疫后一個月,用RH株弓形蟲速殖子腹腔注射小鼠進行攻擊感染,統(tǒng)計學(xué)比較發(fā)現(xiàn)rSAG5B和rSAG1與粗抗原對小鼠的免疫保護作用沒有顯著性差異(P0.05),而均比空白對照組(未免疫組)生存時間長,有顯著性差異(P0.05)。結(jié)論:成功地從弓形蟲RH株基因組中獲取了sag5b基因以及sag1,構(gòu)建了sag5b-pET28a/sag1-pET28a重組質(zhì)粒,并獲得了高效表達(dá);對比RH株基因組與Praugniud株基因組表明,sag5b可能作為鑒別強毒株與弱毒株的遺傳標(biāo)志。制備RH株弓形蟲速殖子粗抗原并免疫新西蘭白兔獲得抗弓形蟲多抗血清。粗抗原與獲得的純化重組蛋白分別免疫Balb/c小鼠,并與空白做比較發(fā)現(xiàn),rSAG5B以及rSAG1均對小鼠有免疫保護作用,且與粗抗原的作用相仿。對弓形蟲新發(fā)現(xiàn)的表膜蛋白SAG5B對小鼠的免疫保護性的研究,有助于開發(fā)有效的預(yù)防弓形蟲感染的疫苗。
[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.

【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】：2008
【分類號】：R392

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