本文關(guān)鍵詞： 人類免疫缺陷病毒 單克隆抗體 膠體金免疫層析 p24檢測 酶聯(lián)免疫吸附測定　出處：《華南理工大學》2013年碩士論文　論文類型：學位論文

【摘要】：P24蛋白是1型人類免疫缺陷病毒(HIV-1)的衣殼蛋白,在病毒的包裝和成熟過程中起著重要的作用。P24蛋白在HIV-1感染者感染早期和晚期的血清中含量相對較高,因此p24蛋白被認為是HIV-1早期檢測、獻血篩查以及監(jiān)測疾病進程的重要指標。 本文根據(jù)數(shù)據(jù)庫中p24蛋白的氨基酸序列,設(shè)計并合成編碼基因且成功構(gòu)建pET28a-p24原核表達質(zhì)粒,成功在大腸桿菌BL21(DE3)中表達并用親和層析純化得到了純度高于95%的重組p24蛋白,間接酶聯(lián)免疫吸附測定(ELISA)結(jié)果表明重組p24蛋白可被抗p24抗原的單克隆抗體識別。使用重組p24蛋白免疫Balb/c小鼠,將免疫后血清效價達到融合要求的小鼠的脾細胞與SP2/0骨髓瘤細胞融合制備雜交瘤細胞。以p24蛋白作為抗原進行間接ELISA法篩選,經(jīng)多次有限稀釋法亞克隆得到33株穩(wěn)定分泌抗p24抗原的抗體的雜交瘤細胞株,制備腹水并用辛酸硫酸銨聯(lián)合沉淀法或Protein G注親和層析法純化單克隆抗體。將得到的單克隆抗體進行ELISA平臺的雙抗體夾心配對,接著將得到的34種能在ELISA平臺配對的抗體組合應(yīng)用于膠體金免疫層析檢測(GICA)平臺的p24抗原檢測,最終篩選出靈敏度和特異性相對最高的單克隆抗體組合3-1D5和4-8A10,該抗體組合在GICA平臺對p24抗原的最低檢測濃度為25pg/mL。利用3-1D5和4-8A10抗體組合在GICA平臺檢測HIV-1p24抗原國家參考品,對其中全部的10個陽性參考品均檢出陽性并可以檢測參考品中濃度低至5U/mL的WHO推薦p24蛋白標準品,表現(xiàn)出較高的靈敏度。再檢測HIV抗原抗體均為陰性的血清樣品(n=153),其中出3份假陽結(jié)果,顯示出了良好的特異性。實驗結(jié)果表明用制備得到的單克隆抗體組合開發(fā)GICA法p24抗原快速檢測有很好的應(yīng)用前景,為HIV第四代快速檢測試劑的研發(fā)打下了堅實的基礎(chǔ)。
[Abstract]:P24 protein is the capsid protein of human immunodeficiency virus type 1 (HIV-1). P24 protein plays an important role in the packaging and maturation of the virus. P24 protein is relatively high in the early and late stage of HIV-1 infection. Therefore, p24 protein is considered to be an important indicator of early detection of HIV-1, screening of blood donors and monitoring of disease progression. According to the amino acid sequence of p24 protein in the database, the encoding gene was designed and synthesized, and the prokaryotic expression plasmid of pET28a-p24 was successfully constructed. The recombinant p24 protein with purity of more than 95% was obtained by affinity chromatography. Indirect enzyme-linked immunosorbent assay (Elisa) showed that the recombinant p24 protein could be recognized by monoclonal antibodies against p24 antigen. Balb/c mice were immunized with recombinant p24 protein. Hybridoma cells were prepared by fusion of spleen cells of mice with SP2/0 myeloma cells whose titer of serum reached fusion requirement after immunization. P24 protein was used as antigen for indirect ELISA screening. 33 hybridoma cell lines secreting antibodies against p24 antigen were obtained by multiple limited dilution subcloning. Preparation of ascites and purification of monoclonal antibodies with ammonium octanoate combined with precipitation or Protein G affinity chromatography. The obtained monoclonal antibodies were paired with double antibodies of ELISA platform. Then, 34 kinds of antibody combinations which can be paired on ELISA platform were used to detect p24 antigen of GICA platform by colloidal gold immunochromatography. Finally, the relative highest sensitivity and specificity of monoclonal antibody combinations 3-1D5 and 4-8A10 were screened. The minimum detection concentration of p24 antigen on GICA platform was 25pg / mL. using the combination of 3-1D5 and 4-8A10 antibodies to detect HIV-1p24 antigen in GICA platform, All of the 10 positive reference samples were positive and could be used to detect p24 protein standard samples with a concentration as low as 5 U / mL in the reference. The serum samples with negative HIV antigen and antibody were all negative, and 3 of them had false positive results. The experimental results show that the rapid detection of p24 antigen by GICA method using the prepared monoclonal antibody combination has a good application prospect and lays a solid foundation for the development of rapid detection reagent for HIV 4th generation.
【學位授予單位】：華南理工大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R512.91;R392

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