细粒棘球蚴（中国大陆株）重组EgP-29抗原的克
发布时间：2018-08-24 11:22
【摘要】： 目的通过分子生物学技术,获取细粒棘球蚴(Echinococcus granulosus, Eg)P-29重组蛋白,进行实验动物免疫保护性及其免疫机制的研究,以鉴定其疫苗的潜质。方法(1)细粒棘球蚴诊断抗原P-29基因(EgP-29)的克隆及序列分析:①以细粒棘球蚴原头蚴RNA为模板,根据互联网GenBank检索出EgP-29基因序列设计引物,采用RT-PCR扩增目的基因;将目的基因克隆到pGEM-T载体,转化入大肠杆菌JM109,对EgP-29基因进行克隆及DNA测序;②应用DNAstar、Biosun生物分析软件对EgP-29蛋白的二级结构、抗原表位、三维结构进行预测,应用NCBI/BLAST公共数据库对目的蛋白进行同源性比较分析;(2)重组表达质粒的构建、表达及免疫学特性鉴定:①将目的基因亚克隆到pET-28a表达载体,转化入大肠杆菌BL21(DE3)plysS,诱导表达重组蛋白EgP-29(rEgP-29),经含Ni2+的His-bind树脂柱纯化rEgP-29;②用rEgP-29免疫小鼠得到抗血清,通过Western-blot及ELISA对rEgP-29的免疫学特性进行研究。(3)rEgP-29诱导小鼠的免疫保护力及其机制研究:①rEgP-29诱导小鼠的免疫保护力观察;②rEgP-29诱导小鼠T淋巴细胞亚群变化的研究;③rEgP-29诱导小鼠细胞因子变化的研究及MTT法检测小鼠脾淋巴细胞增殖情况。结果(1):①通过RT-PCR技术成功扩增目的片段,该基因开放阅读框长度为717bp,构建基因工程菌株EgP-29/pGEM-T/JM109。②EgP-29基因测序结果与GenBank中已发表的基因序列相比同源性为100%,氨基酸序列同源性亦为100%。DANstar软件分析EgP-29分子量约27kDa,极性氨基酸数目68个,转角和不规则卷曲二级结构占21%,Biosun软件分析其抗原表位位点15个。(2):①构建基因工程菌株EgP-29/pET-28a/BL21(DE3)plysS,表达并纯化出分子量约31kD的rEgP-29。②初步鉴定rEgP-29的免疫学特性:Western-blot检测结果显示,rEgP-29免疫小鼠的抗血清可识别rEgP-29、天然抗原原头蚴;细粒棘球蚴感染的兔抗血清也可以识别rEgP-29。ELISA结果显示,免疫组血清中的吸光度值明显高于对照组血清(P0.01)。(3):①rEgP-29能诱导小鼠产生96.6%的免疫保护力,rEgP-29组小鼠在抗原免疫后和攻击感染后均产生高水平的IgG、IgG1和IgG3抗体,低水平的IgG2a和IgE,IgG2b水平在攻击前是升高的,在攻击后是降低的,提示IgG、IgG1、IgG3u可能参与保护性的免疫应答机制。②rEgP-29组小鼠在攻击感染后不仅CD4+T细胞增加,CD8+T细胞也有轻度增加,CD4+/CD8+比值与PBS对照组比较有所降低。③rEgP-29组小鼠在攻击感染后产生高水平的IL-2,低水平的IL-4和IFN-γ,提示该重组抗原以诱导Th1型免疫应答为主的反应;同时攻击感染后进行脾淋巴细胞增殖试验,MTT法检测证实rEgP-29免疫的小鼠在rEgP-29刺激下脾淋巴细胞增殖水平明显高于PBS对照组(P0.01)。结论(1):成功扩增中国大陆株细粒棘球蚴诊断抗原P-29基因,构建基因工程菌株EgP-29/pGEM-T/JM109。EgP-29结构、功能及抗原表位的预测对本实验的实施及选取有价值的抗原肽段提供了理论依据。( 2 ):成功构建基因工程菌株EgP-29/pET-28a/BL21(DE3)plysS,表达并纯化出rEgP-29。经免疫学鉴定初步说明:rEgP-29有较好的抗原性及免疫原性,具有抗包虫病疫苗候选分子的潜能。(3):rEgP-29能诱导小鼠产生部分免疫保护力,其保护性免疫主要通过诱导宿主产生体液免疫应答和Th1型免疫应答,表明rEgP-29是具有发展前途的抗包虫病候选疫苗。
[Abstract]:Objective To obtain the recombinant protein of Echinococcus granulosus (Eg) P-29 by molecular biology technique and to study the immune protective effect and immune mechanism of the vaccine in experimental animals. RNA was used as a template to design primers according to the gene sequence of EgP-29 retrieved from Internet GenBank, and the target gene was amplified by RT-PCR. The target gene was cloned into pGEM-T vector and transformed into E.coli JM109 for cloning and sequencing of EgP-29 gene. DNA star and Biosun were used to analyze the secondary structure, epitope and antigen of EgP-29 protein. Dimensional structure was predicted, and the homology of the target protein was analyzed by NCBI/BLAST public database. (2) Construction, expression and immunological characterization of recombinant expression plasmid: (1) Subcloning the target gene into pET-28a expression vector, transforming it into E.coli BL21 (DE3) plysS, inducing the expression of recombinant protein EgP-29 (rEgP-29) by Ni2+ containing Hei. REgP-29 was purified by s-bind resin column; (2) Antiserum was obtained from mice immunized with rEgP-29, and the immunological characteristics of rEgP-29 were studied by Western-blot and ELISA. (3) Immunoprotective effect of rEgP-29 on mice induced by rEgP-29 and its mechanism: (1) Immunoprotective effect of rEgP-29 on mice induced by rEgP-29; (2) Changes of T lymphocyte subsets induced by rEgP-29 in mice Results: (1) The target fragment was successfully amplified by RT-PCR. The length of the open reading frame of the gene was 717 bp. The sequencing results of EgP-29/pGEM-T/JM109. The molecular weight of EgP-29 was about 27 kDa, the number of polar amino acids was 68, the secondary structure of corner and irregular curl was 21%, and the antigenic epitope sites were 15 by Biosun software. (2) The recombinant strain EgP-29/pET-28a/BL21 (DE3) plysS was constructed, expressed and purified. Preliminary identification of the immunological characteristics of rEgP-29 with molecular weight of about 31 kD: Western-blot results showed that the antisera of rEgP-29 immunized mice could recognize rEgP-29, the natural antigen protocercariae; the rabbit antisera of Echinococcus granulosus infection could also recognize rEgP-29. ELISA results showed that the absorbance value of the sera of the immunized group was significantly higher than that of the control group. Group serum (P 0.01). (3): (1) rEgP-29 could induce 96.6% immune protection in mice. High levels of IgG, IgG1 and IgG3 antibodies were produced in rEgP-29 mice after antigen immunization and attack infection. Low levels of IgG2a, IgE and IgG2b were elevated before attack and decreased after attack, suggesting that IgG, IgG1 and IgG3u might be involved in the protective effect. (2) The immune response mechanism of rEgP-29 mice after attack infection was not only increased CD4+T cells, but also slightly increased CD8+T cells. The ratio of CD4+/CD8+ was lower than that of PBS control group. (3) The rEgP-29 mice produced high levels of IL-2, low levels of IL-4 and IFN-gamma after attack infection, suggesting that the recombinant antigen could induce Th1 type immune response. The proliferation of splenic lymphocytes in mice immunized with rEgP-29 was significantly higher than that in PBS control group (P 0.01). CONCLUSION (1) The P-29 gene of Echinococcus granulosus antigen from mainland China was successfully amplified and the gene engineering strain EgP-29 / pGEM-T / pGEM-T was constructed. The prediction of the structure, function and epitope of JM109.EgP-29 provides a theoretical basis for the implementation of this experiment and the selection of valuable antigenic peptides. (2) The genetic engineering strain EgP-29/pET-28a/BL21 (DE3) plysS was successfully constructed, and rEgP-29 was expressed and purified. Potential of candidate molecules of anti-hydatid vaccine. (3): rEgP-29 can induce partial immune protection in mice. Its protective immunity mainly induces humoral immune response and Th1 immune response of the host, indicating that rEgP-29 is a promising candidate vaccine against hydatid disease.
【学位授予单位】：宁夏医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2200668
[Abstract]:Objective To obtain the recombinant protein of Echinococcus granulosus (Eg) P-29 by molecular biology technique and to study the immune protective effect and immune mechanism of the vaccine in experimental animals. RNA was used as a template to design primers according to the gene sequence of EgP-29 retrieved from Internet GenBank, and the target gene was amplified by RT-PCR. The target gene was cloned into pGEM-T vector and transformed into E.coli JM109 for cloning and sequencing of EgP-29 gene. DNA star and Biosun were used to analyze the secondary structure, epitope and antigen of EgP-29 protein. Dimensional structure was predicted, and the homology of the target protein was analyzed by NCBI/BLAST public database. (2) Construction, expression and immunological characterization of recombinant expression plasmid: (1) Subcloning the target gene into pET-28a expression vector, transforming it into E.coli BL21 (DE3) plysS, inducing the expression of recombinant protein EgP-29 (rEgP-29) by Ni2+ containing Hei. REgP-29 was purified by s-bind resin column; (2) Antiserum was obtained from mice immunized with rEgP-29, and the immunological characteristics of rEgP-29 were studied by Western-blot and ELISA. (3) Immunoprotective effect of rEgP-29 on mice induced by rEgP-29 and its mechanism: (1) Immunoprotective effect of rEgP-29 on mice induced by rEgP-29; (2) Changes of T lymphocyte subsets induced by rEgP-29 in mice Results: (1) The target fragment was successfully amplified by RT-PCR. The length of the open reading frame of the gene was 717 bp. The sequencing results of EgP-29/pGEM-T/JM109. The molecular weight of EgP-29 was about 27 kDa, the number of polar amino acids was 68, the secondary structure of corner and irregular curl was 21%, and the antigenic epitope sites were 15 by Biosun software. (2) The recombinant strain EgP-29/pET-28a/BL21 (DE3) plysS was constructed, expressed and purified. Preliminary identification of the immunological characteristics of rEgP-29 with molecular weight of about 31 kD: Western-blot results showed that the antisera of rEgP-29 immunized mice could recognize rEgP-29, the natural antigen protocercariae; the rabbit antisera of Echinococcus granulosus infection could also recognize rEgP-29. ELISA results showed that the absorbance value of the sera of the immunized group was significantly higher than that of the control group. Group serum (P 0.01). (3): (1) rEgP-29 could induce 96.6% immune protection in mice. High levels of IgG, IgG1 and IgG3 antibodies were produced in rEgP-29 mice after antigen immunization and attack infection. Low levels of IgG2a, IgE and IgG2b were elevated before attack and decreased after attack, suggesting that IgG, IgG1 and IgG3u might be involved in the protective effect. (2) The immune response mechanism of rEgP-29 mice after attack infection was not only increased CD4+T cells, but also slightly increased CD8+T cells. The ratio of CD4+/CD8+ was lower than that of PBS control group. (3) The rEgP-29 mice produced high levels of IL-2, low levels of IL-4 and IFN-gamma after attack infection, suggesting that the recombinant antigen could induce Th1 type immune response. The proliferation of splenic lymphocytes in mice immunized with rEgP-29 was significantly higher than that in PBS control group (P 0.01). CONCLUSION (1) The P-29 gene of Echinococcus granulosus antigen from mainland China was successfully amplified and the gene engineering strain EgP-29 / pGEM-T / pGEM-T was constructed. The prediction of the structure, function and epitope of JM109.EgP-29 provides a theoretical basis for the implementation of this experiment and the selection of valuable antigenic peptides. (2) The genetic engineering strain EgP-29/pET-28a/BL21 (DE3) plysS was successfully constructed, and rEgP-29 was expressed and purified. Potential of candidate molecules of anti-hydatid vaccine. (3): rEgP-29 can induce partial immune protection in mice. Its protective immunity mainly induces humoral immune response and Th1 immune response of the host, indicating that rEgP-29 is a promising candidate vaccine against hydatid disease.
【学位授予单位】：宁夏医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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