本文選題：結(jié)核桿菌　切入點(diǎn)：Hsp65　出處：《華中科技大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:構(gòu)建人結(jié)核桿菌Hsp65和Ag85B膜錨定共表達(dá)質(zhì)粒pIRES-MTHsp65 -Ag85B,并檢測其在體外的表達(dá)。 方法:利用PCR技術(shù)分別從質(zhì)粒pBCG-SP-Hsp65和結(jié)核桿菌H37Rv的基因組中擴(kuò)增出Hsp65和Ag85B基因。并將其克隆到真核表達(dá)質(zhì)粒pVAC中,分別獲得重組質(zhì)粒pVAC-Hsp65、pVAC-Ag85B。然后分別以pVAC-Hsp65、pVAC-Ag85B質(zhì)粒為模板,采用PCR技術(shù)擴(kuò)增出5′端含編碼人白介素2(IL-2)23個(gè)氨基酸的信號(hào)序列與3′端含編碼人胎盤堿性磷酸酶(PLAP)COOH-末端32個(gè)氨基酸的膜錨定序列的Hsp65、Ag85B修飾基因。并將這兩個(gè)修飾基因先后定向克隆到真核表達(dá)載體pIRES上獲得共表達(dá)質(zhì)粒pIRES-MTHsp65-Ag85B。將重組質(zhì)粒轉(zhuǎn)染Hela細(xì)胞,通過RT-PCR的方法檢測轉(zhuǎn)染細(xì)胞中Hsp65和Ag85B mRNA的表達(dá)。 結(jié)果:經(jīng)過PCR、酶切鑒定和測序證實(shí)所克隆的Hsp65、Ag85B基因與報(bào)道結(jié)果完全一致,重組真核表達(dá)質(zhì)粒pIRES-MTHsp65-Ag85B構(gòu)建成功,并且轉(zhuǎn)染Hela細(xì)胞總RNA中結(jié)核桿菌Hsp65和Ag85B mRNA為陽性。 結(jié)論:成功構(gòu)建了人結(jié)核桿菌Hsp65和Ag85B膜錨定雙表達(dá)質(zhì)粒pIRES-MTHsp65-Ag85B,該質(zhì)粒轉(zhuǎn)染人子宮頸癌Hela細(xì)胞后獲得表達(dá),為進(jìn)一步對其免疫原性和抗感染效應(yīng)的研究奠定了基礎(chǔ)。 目的構(gòu)建含日本血吸蟲相對分子質(zhì)量為26 000的抗原(Sj26GST)基因的膜錨定表達(dá)DNA疫苗,觀察其免疫BALB/c小鼠后的免疫應(yīng)答效應(yīng)。 方法用RT-PCR法,以血吸蟲成蟲RNA為模板,擴(kuò)增獲得Sj26的全長基因。利用重組PCR技術(shù),在Sj26基因的5′端加上編碼IL-2的信號(hào)肽核苷酸序列,3′端加上編碼胎盤堿性磷酸酶的膜錨定序列,然后將其克隆入pIRES載體中,構(gòu)建一個(gè)膜錨定型真核表達(dá)質(zhì)粒pIRES-Sj26。將重組質(zhì)粒轉(zhuǎn)染HeLa細(xì)胞,通過RT-PCR及間接免疫熒光技術(shù)檢測目的基因的表達(dá)。用構(gòu)建的pIRES-Sj26疫苗肌肉注射免疫BALB/c小鼠后,用ELISA試劑盒檢測小鼠血清中的總IgG抗體濃度和脾淋巴細(xì)胞培養(yǎng)上清的干擾素γ(INF-γ)含量,以淋巴細(xì)胞刺激指數(shù)(SI)反應(yīng)淋巴細(xì)胞增殖能力,用流式細(xì)胞術(shù)檢測脾細(xì)胞CD4/CD8亞群。 結(jié)果經(jīng)過酶切鑒定、PCR及測序證實(shí)重組質(zhì)粒pIRES-Sj26構(gòu)建成功,經(jīng)轉(zhuǎn)染HeLa細(xì)胞及免疫熒光檢測證明質(zhì)粒pIRES-Sj26能在體外進(jìn)行表達(dá)。免疫小鼠后檢測結(jié)果表明pIRES-Sj26組的血清總IgG抗體濃度、INF-γ的含量明顯高于對照組和空載體組(P0.01);其脾SI高于對照組和空載體組(P0.05);CD8+細(xì)胞百分比高于對照組和空載體組(P0.05), CD4+細(xì)胞百分比沒有顯著變化(P0.05)。 結(jié)論成功構(gòu)建日本血吸蟲膜錨定表達(dá)DNA疫苗pIRES-Sj26,表達(dá)的Sj26蛋白大部分錨定在細(xì)胞膜上。pIRES-Sj26疫苗能增強(qiáng)BALB/c小鼠的免疫應(yīng)答反應(yīng)。
[Abstract]:Aim: to construct the co-expression plasmid pIRES-MTHsp65-Ag85B of Hsp65 and Ag85B, and to detect its expression in vitro. Methods: the Hsp65 and Ag85B genes were amplified by PCR from the genome of plasmid pBCG-SP-Hsp65 and Mycobacterium tuberculosis H37Rv, and cloned into the eukaryotic expression plasmid pVAC. The recombinant plasmid pVAC-Hsp65 pVAC-Ag85B was obtained, and then the plasmid pVAC-Hsp65 pVAC-Ag85B was used as a template. PCR technique was used to amplify the signal sequence of 23 amino acids encoding human interleukin-2 (IL-2) and Hsp65Ag-85B modified gene with 3'terminal mooring sequence encoding 32 amino acids of human placental alkaline phosphatase (PLAPAPP) COOH- terminal. The co-expression plasmid pIRES-MTHsp65-Ag85Bwas cloned into eukaryotic expression vector pIRES. The recombinant plasmid was transfected into Hela cells. The expression of Hsp65 and Ag85B mRNA in transfected cells was detected by RT-PCR. Results: the cloned Hsp65 Ag85B gene was confirmed by PCR, restriction endonuclease digestion and sequencing. The recombinant eukaryotic expression plasmid pIRES-MTHsp65-Ag85B was successfully constructed, and Hsp65 and Ag85B mRNA were positive in the total RNA of Hela cells. Conclusion: the double expression plasmid pIRES-MTHsp65-Ag85B was successfully constructed by Hsp65 and Ag85B membrane anchoring. The plasmid was transfected into human cervical cancer Hela cells and was expressed successfully, which laid a foundation for the further study of its immunogenicity and anti-infection effect. Objective to construct the membrane anchored DNA vaccine containing Schistosoma japonicum antigen Sj26GST gene and observe the immune response of BALB/c mice immunized with Sj26GSTgene. Methods the full-length gene of Sj26 was amplified by RT-PCR and RNA of adult Schistosoma japonicum was used as template, and the recombinant PCR technique was used. The signal peptide nucleotide sequence encoding IL-2 was added to the 5 'terminal of Sj26 gene and the membrane anchoring sequence encoding placental alkaline phosphatase was added to the 3' terminal of IL-2 gene. Then it was cloned into pIRES vector. A membrane anchored eukaryotic expression plasmid pIRES-Sj26 was constructed. The recombinant plasmid was transfected into HeLa cells. The expression of the target gene was detected by RT-PCR and indirect immunofluorescence technique. The BALB/c mice were injected intramuscularly with the constructed pIRES-Sj26 vaccine. The concentration of total IgG antibody in serum and the content of interferon 緯 -INF- 緯 in the supernatant of spleen lymphocytes were detected by ELISA kit. Lymphocyte proliferation was measured by lymphocyte stimulating index (SI). The CD4/CD8 subsets of splenocytes were detected by flow cytometry. Results the recombinant plasmid pIRES-Sj26 was successfully constructed by PCR and sequencing. Transfection of HeLa cells and immunofluorescence assay proved that the plasmid pIRES-Sj26 could be expressed in vitro. The results of immunized mice showed that the serum total IgG antibody concentration of pIRES-Sj26 group was significantly higher than that of control group and empty vector group (P0.01), and the spleen SI of mice was higher than that of control group and empty vector group. The percentage of CD8 cells in control group and empty carrier group was higher than that in control group and empty carrier group, but the percentage of CD4 cells did not change significantly. Conclusion DNA vaccine pIRES-Sj26 was successfully constructed by membrane anchoring of Schistosoma japonicum. The majority of the expressed Sj26 protein was anchored on the membrane. PIRES-Sj26 vaccine could enhance the immune response of BALB/c mice.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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本文編號(hào)：1631079