HA-81-122-Fdc和HA-81-122-Fc融合蛋白制备及免疫学功能检测

科研成果详情

题名	HA-81-122-Fdc和HA-81-122-Fc融合蛋白制备及免疫学功能检测
其他题名	Preparation and Immunologic Function Evaluation ofHA-81-122-Fdc and HA-81-122-Fc Fusion Protein
作者	李叶
学位类型	硕士
导师	高基民
答辩日期	2015-05-30
学位授予单位	温州医科大学
学位专业	临床检验诊断
关键词	流感 H5N1 血球凝集素保守序列粘膜免疫 Fc
摘要	目的在本课题中，我们拟设计了两种融合蛋白（HA-81–122-Fdc 和 HA-81–122-Fc）。融合蛋白由高致病性禽流感（HPAI）H5N1的HA的高保守区（第81-122氨基酸序列）与人IgG 的Fc分子和/或 Folden (Fd) 三聚体模式分子融合而成，从而发展一种以HA1保守序列为基础的能引起粘膜免疫反应的融合蛋白候选疫苗分子。方法1 构建HA-81–122-Fdc 和HA-81–122-Fc重组质粒将病毒株A/Anhui/1/2005(H5N1) 的HA181-122氨基酸残基的碱基序列融合Fd序列是通过用本实验原有的质粒P#295 (HA-3-295-Fdc)作为模板，再用覆盖过Fd的引物进行PCR反应扩增后，获得目的基因片段；然后将该目的基因插入到Pfuse-hIgG1-Fc2表达载体中构建表达HA-81-122-Fdc融合蛋白的重组质粒。HA-81–122-Fc质粒的构建是通过直接将扩增后的目的基因插入到Pfuse-hIgG1-Fc2表达载体中进行构建的。2 HA-81–122-Fdc 和HA-81–122-Fc重组蛋白的真核表达将HA-81–122-Fdc 和HA-81–122-Fc质粒通过磷酸钙的转染方法转染293T细胞。在转染8-10小时后用新鲜的无血清培养基代替原有的细胞培养基，转染72小时后收获细胞上清。用ProteinA亲和柱对含有目的蛋白的细胞上清进行纯化，从而获得纯化的HA-81–122-Fdc 和HA-81–122-Fc融合蛋白。3 用HA-81–122-Fdc 和HA-81–122-Fc融合蛋白免疫实验小鼠将6-8周的雌性BALB/C小鼠分成3组，每组分别5只。将溶在PBS中的融合蛋白与Montanide ISA 51 佐剂按1:1混合，经皮下注射的方法免疫相应组的实验小鼠，首次免疫的蛋白剂量为20ug/只，然后每隔3周进行一次加强免疫，剂量为10ug/只。阴性对照组用同等体积的PBS经皮下注射小鼠。4 ELISA检测免疫小鼠血清和肺灌洗液中的抗体反应本实验用酶联免疫方法ELISA检测免疫小鼠血清和肺灌洗液内的IgG及其亚型的抗体滴度。简述之，先用相应的HA-81-122-Fdc或HA-81-122-Fc融合蛋白包被96孔ELISA板，4°C过夜后，用溶在PBST中的1%的脱脂牛奶对96孔ELISA板进行封闭2h,然后将倍比稀释好的血清加入到相应的ELISA板中，37°孵育1h，然后在用PBST进行洗板4次。洗板过后分别加上相应的HRP-羊抗鼠 IgG (1:2,000稀释), 抗鼠 IgG1 (1:2,000稀释) 或者抗鼠 IgG2a (1:2,000稀释)二抗进行37°C孵育1h。再用PBST洗板4次后加入ELISA显色液TMB，然后用1N H2SO4终止显色反应。将ELISA板放入ELISA读板器中在A450下读板。用ELISA对分泌型IgA的检测与上述的ELISA基本相同，除了肺灌洗液直接加50ul/孔，不需要稀释，而血清则进行1：50的稀释，二抗为HRP- HRP-羊抗鼠 IgA(1:2000稀释)。结果我们的结果显示，所表达的HA-81–122-Fc 和 HA-81–122-Fdc蛋白分别形成了高分子量的二聚体和多聚体，而且都能免疫诱导小鼠产生良好的免疫反应。在其免疫的小鼠血清以及肺洗液内都检测出很强的对HA抗原特异性的IgG抗体反应，血清中抗体滴度达到1:5.2×107。此外，我们还发现，通过加强免疫，HA-81–122-Fdc融合蛋白所引起的IgG1 和 IgG2a 的抗体反应强于HA-81–122-Fc融合蛋白。 HA-81-122融合了Fd和Fc所形成的融合蛋白免疫小鼠后诱导其产生了粘膜免疫反应，小鼠肺灌洗洗液检测到特异的IgA抗体反应。结论将HA蛋白的保守区段与Fc分子融合，或者将HA蛋白的保守区段与形成多聚体形式的分子Fd 融合表达可以成功获得具有多聚体形式的融合蛋白，而且这种融合表达能增强抗原的免疫原性。表达的两种融合蛋白免疫动物后能诱导机体产生良好的体液免疫反应和粘膜免疫反应。这为以此为基础的新型疫苗研究奠定了基础。
其他摘要	Objectives In the present study, we designed two recombinant proteins, HA-81–122-Fdc and HA-81–122-Fc, containing highly conserved HA residues 81–122 of influenza H5N1 fused with foldon (Fd) and/or Fc of human IgG fragment, to develop an HA1 conserved sequence-based mucosal vaccine to prevent H5N1 influenza virus infection. Method1 Construction of recombinant HA-81–122-Fdc and HA-81–122-Fc plasmidThe genes encoding HA1 residues 81–122 of A/Anhui/1/2005(H5N1) (AH/1)[GenBank:ABD28180] fused with Fd were amplified by PCR using our previously constructed HA1-Fdc plasmid [5] and overlapping primers covering Fd as the template and inserted into Pfuse-hIgG1-Fc2 expression vector (hereinafter named Fc, InvivoGen, San Diego, CA) to construct HA-81–122-Fdc recombinant. HA-81–122-Fc was constructed by directly digesting HA-81–122 PCR product and inserting into Fc vector.2 Expression and purification of recombinant HA-81–122-Fdc and HA-81–122-Fc proteinsThe sequence-confirmed recombinant plasmids were transfected into 293T cells (ATCC, Manassas, VA) seeded 24 h prior to transfection, using the calcium phosphate method. Culture medium was replaced by fresh DMEM medium 10 h later, and supernatant was collected 72 h post-transfection. The recombinant HA-81–122-Fdc and HA-81–122-Fc proteins in the supernatant were purified by Protein A affinity chromatography according to the manufacturer’s instructions.3 Mouse vaccination with HA-81-122-Fc and HA-81-122-Fc recombinant proteinsGroups of five female BALB/c mice at 6–8 weeks were respectively subcutaneously (s.c.) primed-vaccinated with 20 µg/mouse of HA-81–122-Fdc and HA-81–122-Fc proteins resuspended in PBS in the presence of Montanide ISA 51 adjuvant and boosted twice with 10 µg/mouse of immunogen containing adjuvant ISA51 at 3-week intervals. Control mice were s.c. injected with the same volume of PBS/ISA 51. 4 ELISA assay for detection of antibody of vaccinated mouse sera and lung washingThe IgG antibody responses and/or subtypes were evaluated by enzyme-linked immunosorbent assay (ELISA) in the collected mouse sera and lung wash flush, as previously described, with some modifications. Briefly, 96-well ELISA plates were precoated, respectively, with recombinant HA-81–122-Fdc and HA-81–122-Fc fusion proteins at 4ºC overnight and blocked with 2% non-fat milk at 37ºC for 2 h. Serially diluted mouse sera and lung wash flush were added to the plates and incubated at 37ºC for 1 h, followed by four washes. Bound antibodies were incubated with HRP-conjugated goat anti-mouse IgG (1:2,000), anti-mouse IgG1 (1:2,000) or anti-mouse IgG2a (1:2,000), respectively, for 1 h at 37ºC. The reaction was visualized by substrate 3,3,5,5-tetramethylbenzidine (TMB) and stopped by 1N H2SO4. The absorbance at 450 nm (A450) was measured by ELISA plate reader.Secretory anti-HA IgA antibody responses in lung wash fluid were measured by ELISA using protocols similar to those described above, except for the addition of lung lavage fluid at 50 µl/well or sera (1:50, 50 µl/well) in duplicate wells of the plates. The HRP-conjugated goat anti-mouse IgA was added at a dilution of 1:2,000, followed by measuring the absorbance at 450 nm (A450).ResultsOur results showed that expressed HA-81–122-Fc and HA-81–122-Fdc proteins formed high molecular weight dimer and oligomer, respectively, that induced potent HA-specific IgG antibody responses in both vaccinated mouse sera and lung wash flush, the average antiboday titer is 1:5.2×107. In addition, stronger IgG1 (Th2-associated) and IgG2 (Th1-associated) antibody responses could be raised in the sera of mice following the last boost vaccination of HA-81–122-Fdc than those raised by HA-81–122-Fc vaccination. Importantly, recombinant HA-81–122 protein fused with Fd and Fc is able to elicit high titers of IgA antibody in vaccinated mouse sera and lung wash flush through the parenteral immunization pathway. ConclusionOur data revealed that recombinant protein containing highly conserved HA residues 81–122 of influenza H5N1 fused with Fd and Fc of human IgG could induce strong local mucosal and systemic humoral immune responses in vaccinated animals, revealing the possibility of developing an effective Fc-mediated mucosal H5N1 vaccine based on the highly conserved HA sequences of influenza H5N1
语种	中文
学号	2010010238
发布年限	2010-05-27
毕业论文分类号	0R04
原始专题	检验医学院暨生命科学院
学位论文研究方向	流感疫苗的研究
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全文文件名	2010010238李叶2013临床检验诊断.pdf\|2010010238李叶2013临床检验诊断.pdf
文献类型	学位论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/116371
专题	温州医科大学
作者单位	溫州医科大学检验医学院暨生命科学院
推荐引用方式 GB/T 7714	李叶. HA-81-122-Fdc和HA-81-122-Fc融合蛋白制备及免疫学功能检测[D]. 温州医科大学,2015.