【摘要】： 目的建立轉(zhuǎn)染人突變CD59(hmCD59)-pALTER-MAX重組質(zhì)粒的CHO細(xì)胞模型；利用ANTI-FLAG M2親和層析的方法提取純化hmCD59重組蛋白；以純化的hmCD59重組蛋白免疫Balb／c小鼠，制備抗hmCD59的多克隆及單克隆抗體；為最終設(shè)計出以抗hmCD59單克隆抗體為基礎(chǔ)的診斷與治療措施打下堅實的理論基礎(chǔ)，為糖尿病血管并發(fā)癥的研究提供新的思路與手段。 方法將含有hmCD59全長序列的重組pALTER-MAX質(zhì)粒，運用脂質(zhì)體介導(dǎo)法，與pcDNA3質(zhì)粒共轉(zhuǎn)染中國倉鼠卵巢細(xì)胞(CHO細(xì)胞)，，以新霉素類似物G418篩選出抗性克��；采用免疫熒光技術(shù)篩選轉(zhuǎn)染細(xì)胞陽性克��；不連續(xù)聚丙烯酰胺凝膠電泳(SDS-PAGE)檢測轉(zhuǎn)染CHO細(xì)胞hmCD59的表達(dá)情況；表達(dá)產(chǎn)物經(jīng)ANTI-FLAG M2 affinity gel親和層析純化，以SDS-PAGE、免疫印跡法(Western blot)和酶聯(lián)免疫吸附實驗(ELISA)對純化產(chǎn)物進(jìn)行鑒定；將純化的hmCD59重組蛋白免疫Balb／c小鼠，分別制備抗hmCD59的多克隆及單克隆抗體，ELISA法檢測抗體效價及免疫學(xué)活性。 結(jié)果免疫熒光與SDS-PAGE檢測證實建立了表達(dá)hmCD59的CHO轉(zhuǎn)染細(xì)胞模型；經(jīng)ANTI-FLAG M2 affinity gel親和層析獲得電泳純的hmCD59，蛋白濃度為0.6mg／ml，Western blot與ELISA檢測結(jié)果顯示純化hmCD59與抗hmCD59抗體特異結(jié)合；純化hmCD59免疫Balb／c小鼠獲得的抗血清間接ELISA效價為1∶100；通過淋巴細(xì)胞雜交瘤技術(shù)獲得1株分泌特異性單克隆抗體的雜交瘤細(xì)胞株，其培養(yǎng)上清效價為1∶16。 結(jié)論成功構(gòu)建了轉(zhuǎn)染hmCD59-pALTER-MAX重組質(zhì)粒的CHO細(xì)胞模型；重組hmCD59在CHO細(xì)胞表面穩(wěn)定表達(dá)；親和層析法可以獲得電泳純并具有免疫學(xué)活性的hmCD59蛋白；進(jìn)而免疫Balb／c小鼠獲得了抗hmCD59的多克隆及單克隆抗體，為抗hmCD59抗體的功能研究及臨床應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Objective to establish a CHO cell model transfected with human mutant CD59 (hmCD59) -pALTER-MAX recombinant plasmid, extract and purify hmCD59 recombinant protein by ANTI-FLAG M2 affinity chromatography, immunize Balb/c mice with purified hmCD59 recombinant protein, and prepare anti-hmCD59 polyclonal and monoclonal antibodies. It provides a solid theoretical basis for the design of diagnostic and therapeutic measures based on monoclonal antibodies against hmCD59, and provides new ideas and means for the study of diabetic vascular complications. Methods Recombinant pALTER-MAX plasmid containing the full-length hmCD59 sequence was co-transfected with pcDNA3 plasmid into Chinese hamster ovarian cells (CHO cells) by liposome mediated method. The resistant clones were screened by neomycin analogues G418. The positive clones were screened by immunofluorescence technique, the expression of hmCD59 in transfected CHO cells was detected by discontinuous polyacrylamide gel electrophoresis (SDS-PAGE), and the expression product was purified by ANTI-FLAG M2 affinity gel affinity chromatography. SDS-PAGE, immunoblot assay (Western blot) and enzyme-linked immunosorbent assay (ELISA) were used to identify the purified product, and the purified hmCD59 recombinant protein was used to immunize Balb/c mice. The polyclonal anti-hmCD59 and monoclonal antibody were prepared to detect the antibody titer and immunological activity by Elisa. Results the CHO transfected cell model expressing hmCD59 was established by immunofluorescence and SDS-PAGE detection, and the concentration of hmCD59, protein was 0.6 mg / ml ~ (-1) by ANTI-FLAG M2 affinity gel affinity chromatography. The results showed that the purified hmCD59 specifically combined with anti-hmCD59 antibody. The indirect antiserum ELISA titer of purified hmCD59 immunized Balb/c mice was 1: 100, and a hybridoma cell line secreting specific monoclonal antibody was obtained by lymphocyte hybridoma technique, the titer of culture supernatant was 1: 16. Conclusion the CHO cell model transfected with hmCD59-pALTER-MAX recombinant plasmid was successfully constructed, the recombinant hmCD59 was stably expressed on the surface of CHO cells, and the purified hmCD59 protein with immunological activity could be obtained by affinity chromatography. The polyclonal and monoclonal antibodies against hmCD59 were obtained by immunizing Balb/c mice, which laid a foundation for the functional study and clinical application of anti hmCD59 antibodies.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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