【摘要】： 目的:構(gòu)建日本血吸蟲Sj14FABP和Sj26GST膜錨定共表達(dá)質(zhì)粒pIRES-Sj26-Sj14,并檢測(cè)其在體外的表達(dá)。 方法:利用RT-PCR法,以日本血吸蟲成蟲總RNA為模板,擴(kuò)增獲得Sj14FABP與Sj26GST的全長基因。并先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-Sj14、pVAC-Sj26。然后分別以pVAC-Sj14、pVAC-Sj26質(zhì)粒為模板,采用PCR技術(shù)擴(kuò)增出含人白介素2(IL-2 )23個(gè)氨基酸的信號(hào)肽與人胎盤堿性磷酸酶(PLAP)COOH-末端32個(gè)氨基酸的膜錨定序列在內(nèi)的Sj14、Sj26修飾基因。并將該兩個(gè)修飾基因共同構(gòu)建到真核表達(dá)載體pIRES上獲得共表達(dá)質(zhì)粒pIRES-Sj26-Sj14,將重組質(zhì)粒轉(zhuǎn)染Hela細(xì)胞,通過RT-PCR的方法及間接免疫熒光技術(shù)檢測(cè)Sj26, Sj14基因的膜錨定表達(dá)。 結(jié)果:經(jīng)過酶切鑒定、PCR、測(cè)序證實(shí)所克隆的Sj14FABP、Sj26GST基因與報(bào)道結(jié)果完全一致,重組真核表達(dá)質(zhì)粒pVAC-Sj14、pVAC-Sj26、pIRES-Sj26-Sj14構(gòu)建成功。并且pIRES-Sj26-Sj14質(zhì)粒在體外轉(zhuǎn)染Hela細(xì)胞后可表達(dá)膜錨定蛋白Sj14與Sj26。 結(jié)論:成功構(gòu)建了日本血吸蟲Sj14FABP和Sj26GST膜錨定雙表達(dá)質(zhì)粒,該質(zhì)粒轉(zhuǎn)染人子宮頸癌Hela細(xì)胞后可正常表達(dá)這兩種蛋白,為下步對(duì)其免疫原性免疫反應(yīng)性及免疫保護(hù)作用的進(jìn)一步的研究奠定了基礎(chǔ),也給血吸蟲疫苗的研究拓寬了思路。
[Abstract]:Aim: to construct the co-expression plasmid pIRES-Sj26-Sj14, of Schistosoma japonicum Sj14FABP and Sj26GST and to detect its expression in vitro. Methods: the full-length genes of Sj14FABP and Sj26GST were amplified by using total RNA of adult Schistosoma japonicum as template by RT-PCR method. The recombinant plasmid pVAC-Sj14,pVAC-Sj26. was obtained by cloning it into eukaryotic expression plasmid pVAC. Then the pVAC-Sj14,pVAC-Sj26 plasmids were used as templates, The Sj14,Sj26 modified genes including the signal peptide containing 23 amino acids of human interleukin 2 (IL-2) and the membrane anchoring sequence of 32 amino acids of (PLAP) COOH- terminal of human placental alkaline phosphatase (PLAP) COOH-) were amplified by PCR. The two modified genes were co-constructed into eukaryotic expression vector pIRES to obtain the co-expression plasmid pIRES-Sj26-Sj14,. The recombinant plasmid was transfected into Hela cells. The membrane anchoring expression of Sj26, Sj14 gene was detected by RT-PCR and indirect immunofluorescence technique. Results: PCR, sequencing confirmed that the cloned Sj14FABP,Sj26GST gene was in good agreement with the reported results. The recombinant eukaryotic expression plasmid pVAC-Sj14,pVAC-Sj26,pIRES-Sj26-Sj14 was successfully constructed. Moreover, pIRES-Sj26-Sj14 plasmid can express membrane anchoring protein Sj14 and Sj26. after transfection of Hela cells in vitro. Conclusion: the double expression plasmids of Schistosoma japonicum Sj14FABP and Sj26GST membrane anchoring have been constructed successfully. These two proteins can be expressed normally after transfected into human cervical cancer Hela cells, which is the next step to identify the immunogenicity of Schistosoma japonicum and Schistosoma japonicum. The further study of immunoreactivity and immune protection has laid a foundation for the research of Schistosoma japonicum vaccine.
【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】：2006
【分類號(hào)】：R392

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