細粒棘球蚴（中國大陸株）重組EgP-29抗原的克
發(fā)布時間：2018-08-24 11:22
【摘要】： 目的通過分子生物學技術,獲取細粒棘球蚴(Echinococcus granulosus, Eg)P-29重組蛋白,進行實驗動物免疫保護性及其免疫機制的研究,以鑒定其疫苗的潛質(zhì)。方法(1)細粒棘球蚴診斷抗原P-29基因(EgP-29)的克隆及序列分析:①以細粒棘球蚴原頭蚴RNA為模板,根據(jù)互聯(lián)網(wǎng)GenBank檢索出EgP-29基因序列設計引物,采用RT-PCR擴增目的基因;將目的基因克隆到pGEM-T載體,轉(zhuǎn)化入大腸桿菌JM109,對EgP-29基因進行克隆及DNA測序;②應用DNAstar、Biosun生物分析軟件對EgP-29蛋白的二級結構、抗原表位、三維結構進行預測,應用NCBI/BLAST公共數(shù)據(jù)庫對目的蛋白進行同源性比較分析;(2)重組表達質(zhì)粒的構建、表達及免疫學特性鑒定:①將目的基因亞克隆到pET-28a表達載體,轉(zhuǎn)化入大腸桿菌BL21(DE3)plysS,誘導表達重組蛋白EgP-29(rEgP-29),經(jīng)含Ni2+的His-bind樹脂柱純化rEgP-29;②用rEgP-29免疫小鼠得到抗血清,通過Western-blot及ELISA對rEgP-29的免疫學特性進行研究。(3)rEgP-29誘導小鼠的免疫保護力及其機制研究:①rEgP-29誘導小鼠的免疫保護力觀察;②rEgP-29誘導小鼠T淋巴細胞亞群變化的研究;③rEgP-29誘導小鼠細胞因子變化的研究及MTT法檢測小鼠脾淋巴細胞增殖情況。結果(1):①通過RT-PCR技術成功擴增目的片段,該基因開放閱讀框長度為717bp,構建基因工程菌株EgP-29/pGEM-T/JM109。②EgP-29基因測序結果與GenBank中已發(fā)表的基因序列相比同源性為100%,氨基酸序列同源性亦為100%。DANstar軟件分析EgP-29分子量約27kDa,極性氨基酸數(shù)目68個,轉(zhuǎn)角和不規(guī)則卷曲二級結構占21%,Biosun軟件分析其抗原表位位點15個。(2):①構建基因工程菌株EgP-29/pET-28a/BL21(DE3)plysS,表達并純化出分子量約31kD的rEgP-29。②初步鑒定rEgP-29的免疫學特性:Western-blot檢測結果顯示,rEgP-29免疫小鼠的抗血清可識別rEgP-29、天然抗原原頭蚴;細粒棘球蚴感染的兔抗血清也可以識別rEgP-29。ELISA結果顯示,免疫組血清中的吸光度值明顯高于對照組血清(P0.01)。(3):①rEgP-29能誘導小鼠產(chǎn)生96.6%的免疫保護力,rEgP-29組小鼠在抗原免疫后和攻擊感染后均產(chǎn)生高水平的IgG、IgG1和IgG3抗體,低水平的IgG2a和IgE,IgG2b水平在攻擊前是升高的,在攻擊后是降低的,提示IgG、IgG1、IgG3u可能參與保護性的免疫應答機制。②rEgP-29組小鼠在攻擊感染后不僅CD4+T細胞增加,CD8+T細胞也有輕度增加,CD4+/CD8+比值與PBS對照組比較有所降低。③rEgP-29組小鼠在攻擊感染后產(chǎn)生高水平的IL-2,低水平的IL-4和IFN-γ,提示該重組抗原以誘導Th1型免疫應答為主的反應;同時攻擊感染后進行脾淋巴細胞增殖試驗,MTT法檢測證實rEgP-29免疫的小鼠在rEgP-29刺激下脾淋巴細胞增殖水平明顯高于PBS對照組(P0.01)。結論(1):成功擴增中國大陸株細粒棘球蚴診斷抗原P-29基因,構建基因工程菌株EgP-29/pGEM-T/JM109。EgP-29結構、功能及抗原表位的預測對本實驗的實施及選取有價值的抗原肽段提供了理論依據(jù)。( 2 ):成功構建基因工程菌株EgP-29/pET-28a/BL21(DE3)plysS,表達并純化出rEgP-29。經(jīng)免疫學鑒定初步說明:rEgP-29有較好的抗原性及免疫原性,具有抗包蟲病疫苗候選分子的潛能。(3):rEgP-29能誘導小鼠產(chǎn)生部分免疫保護力,其保護性免疫主要通過誘導宿主產(chǎn)生體液免疫應答和Th1型免疫應答,表明rEgP-29是具有發(fā)展前途的抗包蟲病候選疫苗。
[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.
【學位授予單位】：寧夏醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
本文編號：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.
【學位授予單位】：寧夏醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
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