本文選題：白紋伊蚊 + 過(guò)敏原��； 參考：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：目的蚊子唾液中的變應(yīng)原能夠誘發(fā)人發(fā)生過(guò)敏反應(yīng)。白紋伊蚊(Aedes albopitus)的分布幾乎遍布全球,其唾液當(dāng)中至少含有16種變應(yīng)原,其變應(yīng)原種類(lèi)是同類(lèi)中最多的。目前,研究證實(shí)埃及伊蚊(Aedes aegypti)的三磷酸腺苷雙磷酸酶(apyrase)是蚊子的最主要交叉變應(yīng)原,并已經(jīng)對(duì)其重組表達(dá),然而對(duì)白紋伊蚊的apyrase蛋白尚未深入研究。本課題擴(kuò)增白紋伊蚊唾液變應(yīng)原apyrase蛋白的編碼基因,在大腸桿菌中進(jìn)行表達(dá),獲得有生物活性的重組apyrase蛋白,運(yùn)用雜交瘤技術(shù)制備抗apyrase單克隆抗體,將為蚊子引起的過(guò)敏性疾病的診斷與治療奠定基礎(chǔ)。 方法選取未吸血的白紋伊蚊雌蚊約40只,提取總RNA,采用RT-PCR擴(kuò)增得到總cDNA;以總cDNA為模板擴(kuò)增apyrase蛋白編碼基因,經(jīng)測(cè)序鑒定后連入載體pET-19b,構(gòu)建原核表達(dá)載體；將載體轉(zhuǎn)入大腸桿菌BL21(DE3) pLysS感受態(tài)細(xì)胞,以異丙基-p-D-硫代半乳糖苷(Isopropy1β-D-Thiogalactoside, IPTG)誘導(dǎo)表達(dá),利用融合蛋白N端存在的6個(gè)組氨酸標(biāo)簽,在變性條件下,通過(guò)Ni-NTA柱純化apyrase蛋白后,以尿素梯度透析的方法且在谷胱甘肽氧化還原體系存在下進(jìn)行復(fù)性。以純化復(fù)性的apyrase蛋白免疫Balb/c小鼠,小鼠產(chǎn)生相應(yīng)抗體后,將其脾細(xì)胞與骨髓瘤細(xì)胞進(jìn)行雜交瘤融合,并通過(guò)間接ELISA法進(jìn)行篩選,有限稀釋法進(jìn)行克隆,獲得分泌抗apyrase單抗的特異性細(xì)胞株,并對(duì)分泌的抗體進(jìn)行亞型測(cè)定。利用斑點(diǎn)印跡試驗(yàn)(Dot blotting),免疫印跡試驗(yàn)(Western blotting)及間接熒光免疫試驗(yàn)(IFA)檢測(cè)重組apyrase蛋白和天然apyrase蛋白的一致性。皮膚敏感試驗(yàn)檢測(cè)重組apyrase蛋白的致敏性。以孔雀石綠顯色法和血小板凝集試驗(yàn)檢測(cè)重組apyrase蛋白的酶活性。 結(jié)果以白紋伊蚊總cDNA中擴(kuò)增出的apyrase蛋白編碼基因,與GenBank中推測(cè)的白紋伊蚊apyrase編碼基因序列有96%的同源性；與埃及伊蚊apyrase編碼基因序列有83%的同源性。經(jīng)酶切鑒定及基因測(cè)序證實(shí),pET19b-apyrase構(gòu)建成功。以重組質(zhì)粒轉(zhuǎn)化BL21(DE3) pLysS菌,以IPTG誘導(dǎo)其表達(dá),進(jìn)行SDS-PAGE分析,結(jié)果顯示其分子量約為60kDa。表達(dá)蛋白主要以包涵體的形式存在于沉淀中,經(jīng)純化復(fù)性后免疫老鼠。雜交瘤融合技術(shù)獲得了能穩(wěn)定分泌抗白紋伊蚊apyrase蛋白的雜交瘤細(xì)胞株,命名1A1A4,1A1D4,1A1H4,2D9D9,2G11C5,2G11B4,6G8F9均為重鏈為IgG1型,輕鏈為κ型。Dot blotting實(shí)驗(yàn)顯示重組apyrase蛋白和白紋伊蚊唾液腺粗提液均可以與抗重組apyrase蛋白的單克隆抗體1A1H4(Mabs)發(fā)生抗原抗體反應(yīng),而與正常鼠血清呈陰性反應(yīng)；Western blotting實(shí)驗(yàn)顯示唾液腺粗提液中天然apyrase蛋白的條帶(60kd)可以與Mabs特異性結(jié)合,唾液腺粗提液中其它蛋白條帶則與Mabs無(wú)反應(yīng),而唾液腺粗提液也與正常鼠血清無(wú)反應(yīng)。間接免疫熒光實(shí)驗(yàn)顯示與Mabs孵育后的冰凍切片唾液腺部位出現(xiàn)亮綠色,為陽(yáng)性反應(yīng),定位天然apyrase蛋白在蚊子唾液腺中的分布。皮膚點(diǎn)刺試驗(yàn)證明重組apyrase能夠誘發(fā)皮膚發(fā)生速發(fā)型和遲發(fā)型過(guò)敏反應(yīng)。以孔雀石綠顯色法測(cè)得重組apyrase蛋白水解ATP和ADP的的酶動(dòng)力學(xué)參數(shù)Km分別為11.6gM和49.5gM,Vmax分別為1.02nM/S/μg和0.81nM/S/μg蛋白。血小板凝集試驗(yàn)證明以PBS作為空白對(duì)照,0.4μM和0.8μM重組apyrase蛋白分別能夠抑制6%和9.5%的依賴(lài)ADP的血小板凝集。 結(jié)論成功擴(kuò)增和克隆白紋伊蚊apyrase蛋白的編碼基因,并在大腸桿菌中進(jìn)行高效表達(dá),制備出了和天然apyrase蛋白有很好一致性的有生物學(xué)活性的重組apyrase蛋白,獲得了穩(wěn)定分泌抗白紋伊蚊apyrase蛋白的雜交瘤細(xì)胞株,為白紋伊蚊所致的過(guò)敏性疾病的診斷和治療奠定了基礎(chǔ)。
[Abstract]:Objective allergen in mosquito saliva can induce anaphylaxis. The distribution of Aedes albopitus is almost all over the world, with at least 16 kinds of allergens in its saliva. Its allergens are the largest in the same species. At present, the study confirms that the Aedes aegypti of Aedes aegypti (apyrase) is an mosquito (apyrase). The apyrase protein of Aedes albopictus has not been deeply studied. This subject amplified the encoding gene of the albopictus albopictus apyrase protein, expressed it in Escherichia coli, obtained the recombinant apyrase protein with bioactivity, and prepared the anti apyrase by hybridoma technique. Monoclonal antibodies will lay the foundation for the diagnosis and treatment of allergic diseases caused by mosquitoes.
Methods a total of 40 female Aedes albopictus (Aedes albopictus) were selected, total RNA was extracted and the total cDNA was amplified by RT-PCR. The apyrase protein encoding gene was amplified by the total cDNA template. After sequencing, it was linked to the carrier pET-19b and constructed the prokaryotic expression vector. The vector was transferred into the BL21 (DE3) pLysS receptive cell of Escherichia coli, and the isopropyl -p-D- thiopedic galactose was used. Isopropy1 beta -D-Thiogalactoside (IPTG) is inducible and uses 6 histidine tags that exist in the N terminal of the fusion protein. After denaturing the apyrase protein by Ni-NTA column, the refolding of the glutathione redox system is carried out by the method of urea gradient dialysis and in the presence of the glutathione redox system. In order to purify the immune Balb/c of the refolding apyrase protein, the immune Balb/c is small. Mice, mice produced corresponding antibodies, the spleen cells and myeloma cells of hybridoma fusion, and screened by indirect ELISA method, the finite dilution method was cloned to obtain the specific cell lines secreting anti apyrase monoclonal antibody, and the secreted antibody was subtype, and the dot blot test (Dot blotting) and Western blot test were used. Western blotting) and indirect immunofluorescence test (IFA) were used to detect the consistency of recombinant apyrase protein and natural apyrase protein. Skin sensitivity test was used to detect the sensitivities of recombinant apyrase protein. The enzyme activity of recombinant apyrase protein was detected by malachite green colorimetric assay and platelet aggregation test.
Results the apyrase protein encoding gene amplified from the total cDNA of Aedes albopictus was 96% homologous to the sequence of the apyrase encoding gene of Aedes albopictus in GenBank, and 83% of the apyrase encoding gene sequence of Aedes aegypti. By enzyme digestion and gene sequencing, the construction of pET19b-apyrase was successful. The recombinant plasmid was transformed into BL21 (D). E3) pLysS bacteria, induced by IPTG, and SDS-PAGE analysis, the results show that the molecular weight of 60kDa. expression protein is mainly in the form of inclusion body in the form of inclusion body in the precipitate, after purified and refolding immune mice. Hybridoma fusion technique has obtained a stable secretion of hybridoma cell line that can stabilize the apyrase protein of Aedes albopictus, named 1A1A4,1A1D4,1A1H The heavy chain of 4,2D9D9,2G11C5,2G11B4,6G8F9 was IgG1, and the light chain was kappa type.Dot blotting experiment showed that the recombinant apyrase protein and the crude extract of the salivary gland of Aedes albopictus all could react with the monoclonal antibody 1A1H4 (Mabs) against the recombinant apyrase protein, but negative reaction with the normal rat blood, and Western blotting experiment showed saliva. The strip of natural apyrase protein (60KD) in the crude extract of the liquid gland could be specifically associated with the Mabs. The other protein bands in the salivary gland crude extract did not react with the Mabs, and the salivary gland crude extracts had no response to the normal rat serum. The indirect immunofluorescence experiment showed that the salivary glands of the frozen section of the salivary gland appeared bright green and positive reaction after incubation with Mabs. The distribution of natural apyrase protein in the salivary glands of mosquitoes. The skin prick test showed that the recombinant apyrase could induce the rapid and delayed allergic reaction of the skin. The kinetic parameters Km of the recombinant apyrase protein hydrolyzed ATP and ADP by the malachite green colorimetric method were 11.6gM and 49.5gM respectively, Vmax was 1.02nM/S/ mu g and 0.81, respectively. NM/S/ micron G protein. Platelet agglutination test showed that PBS as a blank control, 0.4 M and 0.8 M recombinant apyrase protein could inhibit the platelet aggregation of 6% and 9.5% dependent ADP respectively.
Conclusion the encoding gene of Aedes albopictus apyrase protein was amplified and cloned successfully and highly expressed in Escherichia coli. The recombinant apyrase protein, which had good consistency with the natural apyrase protein, was prepared. The hybridoma cell line that secreted the apyrase protein of Aedes albopictus was obtained, which was caused by Aedes albopictus. The diagnosis and treatment of allergic diseases have laid the foundation.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R384.1;Q78

【共引文獻(xiàn)】

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