白纹伊蚊唾液三磷酸腺苷双磷酸酶apyrase的克

发布时间：2018-07-02 22:42

本文选题：白纹伊蚊 + 过敏原　；参考：《复旦大学》2011年硕士论文

【摘要】：目的蚊子唾液中的变应原能够诱发人发生过敏反应。白纹伊蚊(Aedes albopitus)的分布几乎遍布全球,其唾液当中至少含有16种变应原,其变应原种类是同类中最多的。目前,研究证实埃及伊蚊(Aedes aegypti)的三磷酸腺苷双磷酸酶(apyrase)是蚊子的最主要交叉变应原,并已经对其重组表达,然而对白纹伊蚊的apyrase蛋白尚未深入研究。本课题扩增白纹伊蚊唾液变应原apyrase蛋白的编码基因,在大肠杆菌中进行表达,获得有生物活性的重组apyrase蛋白,运用杂交瘤技术制备抗apyrase单克隆抗体,将为蚊子引起的过敏性疾病的诊断与治疗奠定基础。方法选取未吸血的白纹伊蚊雌蚊约40只,提取总RNA,采用RT-PCR扩增得到总cDNA;以总cDNA为模板扩增apyrase蛋白编码基因,经测序鉴定后连入载体pET-19b,构建原核表达载体；将载体转入大肠杆菌BL21(DE3) pLysS感受态细胞,以异丙基-p-D-硫代半乳糖苷(Isopropy1β-D-Thiogalactoside, IPTG)诱导表达,利用融合蛋白N端存在的6个组氨酸标签,在变性条件下,通过Ni-NTA柱纯化apyrase蛋白后,以尿素梯度透析的方法且在谷胱甘肽氧化还原体系存在下进行复性。以纯化复性的apyrase蛋白免疫Balb/c小鼠,小鼠产生相应抗体后,将其脾细胞与骨髓瘤细胞进行杂交瘤融合,并通过间接ELISA法进行筛选,有限稀释法进行克隆,获得分泌抗apyrase单抗的特异性细胞株,并对分泌的抗体进行亚型测定。利用斑点印迹试验(Dot blotting),免疫印迹试验(Western blotting)及间接荧光免疫试验(IFA)检测重组apyrase蛋白和天然apyrase蛋白的一致性。皮肤敏感试验检测重组apyrase蛋白的致敏性。以孔雀石绿显色法和血小板凝集试验检测重组apyrase蛋白的酶活性。结果以白纹伊蚊总cDNA中扩增出的apyrase蛋白编码基因,与GenBank中推测的白纹伊蚊apyrase编码基因序列有96%的同源性；与埃及伊蚊apyrase编码基因序列有83%的同源性。经酶切鉴定及基因测序证实,pET19b-apyrase构建成功。以重组质粒转化BL21(DE3) pLysS菌,以IPTG诱导其表达,进行SDS-PAGE分析,结果显示其分子量约为60kDa。表达蛋白主要以包涵体的形式存在于沉淀中,经纯化复性后免疫老鼠。杂交瘤融合技术获得了能稳定分泌抗白纹伊蚊apyrase蛋白的杂交瘤细胞株,命名1A1A4,1A1D4,1A1H4,2D9D9,2G11C5,2G11B4,6G8F9均为重链为IgG1型,轻链为κ型。Dot blotting实验显示重组apyrase蛋白和白纹伊蚊唾液腺粗提液均可以与抗重组apyrase蛋白的单克隆抗体1A1H4(Mabs)发生抗原抗体反应,而与正常鼠血清呈阴性反应；Western blotting实验显示唾液腺粗提液中天然apyrase蛋白的条带(60kd)可以与Mabs特异性结合,唾液腺粗提液中其它蛋白条带则与Mabs无反应,而唾液腺粗提液也与正常鼠血清无反应。间接免疫荧光实验显示与Mabs孵育后的冰冻切片唾液腺部位出现亮绿色,为阳性反应,定位天然apyrase蛋白在蚊子唾液腺中的分布。皮肤点刺试验证明重组apyrase能够诱发皮肤发生速发型和迟发型过敏反应。以孔雀石绿显色法测得重组apyrase蛋白水解ATP和ADP的的酶动力学参数Km分别为11.6gM和49.5gM,Vmax分别为1.02nM/S/μg和0.81nM/S/μg蛋白。血小板凝集试验证明以PBS作为空白对照,0.4μM和0.8μM重组apyrase蛋白分别能够抑制6%和9.5%的依赖ADP的血小板凝集。结论成功扩增和克隆白纹伊蚊apyrase蛋白的编码基因,并在大肠杆菌中进行高效表达,制备出了和天然apyrase蛋白有很好一致性的有生物学活性的重组apyrase蛋白,获得了稳定分泌抗白纹伊蚊apyrase蛋白的杂交瘤细胞株,为白纹伊蚊所致的过敏性疾病的诊断和治疗奠定了基础。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R384.1;Q78

【共引文献】