猪A类清道夫受体SRA和MARCO的克

发布时间：2018-05-03 05:36

本文选题：清道夫受体 + 原核表达　；参考：《石河子大学》2015年硕士论文

【摘要】：清道夫受体,作为一类先天性免疫受体,在宿主先天性防御中起着重要的作用。然而,到目前为止,对猪清道夫受体的研究还非常有限。目的:为了深入了解猪清道夫受体SRA和MARCO的生物学特性,揭示SRA和MARCO在免疫中的作用,本文以猪A类清道夫受体SRA和MARCO为研究对象,利用原核表达系统对受体的胞外区域进行表达,制备纯化的重组蛋白,以重组蛋白为免疫原,制备多抗血清,验证多抗血清的特异性。此外,从临床发病猪的肺部分离细菌,构建猪SRA或MARCO的稳定细胞系,检测SRA和MARCO介导对细菌的吞噬功能,揭示其生物学功能。材料与方法:①猪SRA和MARCO的分子克隆、表达、纯化及其抗体的制备:根据GenBank SRA(NM_001243874)和MARCO(JQ25702.1)的全基因序列,设计引物,利用猪肺组织扩增SRA和MARCO编码序列片段,克隆至载体pMD18-T中,测序验证扩增序列的正确性。选取SRA胞外区基因片段(711bp)和MARCO胞外区基因片段(709bp),利用PCR技术获得目的片段,分别克隆入原核表达载体pET-28a中,构建重组原核表达载体pET-SRA-c和pET-MARCO-c。利用原核表达系统表达重组蛋白rSRA-c(约32kDa)和rMARCO-c(约31kDa),以亲和层析技术获得纯化的rSRA-c和rMARCO-c。分别利用纯化的rSRA-c和rMARCO-c为免疫原,免疫BALB/c小鼠(100ug/小鼠),制备多抗血清。②SRA和MARCO的真核表达载体及其稳定细胞系的构建:将编码序列分别克隆入PCDNA3.O载体中,构建真核表达质粒,将真核表达质粒转染至CHO细胞中,通过96孔板细胞传代及G418的药物筛选构建稳定表达猪SRA或MARCO的CHO细胞的细胞系,通过RT-PCR和Western blot对细胞系进行验证。?SRA和MARCO介导对细菌吞噬功能的分析:对临床发病猪进行解剖,并从肺部分离细菌并用FITC进行标记,将标记好的细菌与构建的稳定细胞系相互作用,从而对SRA和MARCO的功能进行分析。结果:①扩增结果显示SRA和MARCO的片段大小分别为1341bp和1188bp,与预期结果一致,测序结果与原编码序列相同,表明扩增的序列正确。原核质粒PCR验证结果显示SRA和MARCO的片段大小分别为711bp和709bp,与目的片段大小一致。测序结果与选择的胞外段序列一致,表明原核表达质粒构建成功。经Western blot分析,SRA和MARCO分别在32kDa和31kD左右处出现了条带,与目的片段大小相同,表明实验中制备的多抗血清可以识别rSRA-c(约32kDa)和rMARCO-c(约31kDa),具有很好的免疫原性。②真核质粒PCR结果中SRA和MARCO的大小分别为1341bp和1188bp,与编码序列大小一致,测序结果与编码序列相同,表明真核表达质粒构建成功。RT-PCR结果显示SRA细胞系出现了大小为1341bp的条带,与SRA的编码序列大小相同,MARCO细胞系中出现了片段大小为1188bp的条带,与MARCO的编码序列大小相同。细胞系蛋白的Western blot结果显示SRA的阳性克隆细胞蛋白样在55kD处出现了目的条带,MARCO的阳性克隆细胞蛋白样在小于55kD处出现了目的条带,表明稳定细胞系构建成功。③从临床发病猪的肺中分离获得了胸膜肺炎放线杆菌、大肠杆菌、金黄色葡萄球菌及化脓链球菌。经对猪SRA和MARCO介导对细菌吞噬功能的分析,表明猪SRA和MARCO都能有效地介导对大肠杆菌、金黄色葡萄球菌以及化脓链球菌的吞噬,但都不能介导对胸膜肺炎放线杆菌的吞噬,说明猪SRA和MARCO能够介导对部分细菌的吞噬。本研究不仅为进一步研究猪SRA和MARCO在病原微生物感染中的作用做好铺垫,而且为揭示猪SRA和MARCO在病原菌中的作用奠定基础。
[Abstract]:The scavenger receptor, as a class of congenital immune receptors, plays an important role in the host's congenital defense. However, so far, the study of the pig scavenger receptor is still very limited. Objective: to understand the biological characteristics of SRA and MARCO of the pig scavenger receptor and reveal the role of SRA and MARCO in the immunization. DF receptor SRA and MARCO are used to express the extracellular domain of the receptor by the prokaryotic expression system to prepare the purified recombinant protein. The recombinant protein is used as immunogen to prepare polyanti sera and to verify the specificity of the polyanti sera. In addition, the stable cell lines of porcine SRA or MARCO are constructed from the lung part of the clinically infected pig, and S is constructed to detect S. RA and MARCO mediated the phagocytosis of bacteria and revealed their biological functions. Materials and methods: (1) molecular cloning, expression, purification and antibody preparation of porcine SRA and MARCO: primers were designed based on the whole gene sequence of GenBank SRA (NM_001243874) and MARCO (JQ25702.1), and the amplification of SRA and MARCO coding sequences by pig lung tissue was cloned. In body pMD18-T, the sequence was verified by sequencing, and the SRA extracellular domain gene fragment (711bp) and MARCO extracellular domain gene fragment (709bp) were selected. The target fragments were obtained by PCR technology and were cloned into the prokaryotic expression vector pET-28a respectively. The Recombinant Prokaryotic expression vector pET-SRA-c and pET-MARCO-c. were used to express the recombinant protein by using the prokaryotic expression system. RSRA-c (about 32kDa) and rMARCO-c (about 31kDa), the purified rSRA-c and rMARCO-c. were purified by affinity chromatography, using purified rSRA-c and rMARCO-c as immunogens, immunized BALB/c mice (100ug/ mice), and prepared polyanti sera. (2) the eukaryotic expression vector of SRA and MARCO and the construction of stable cell lines. In the carrier, the eukaryotic expression plasmid was constructed and the eukaryotic expression plasmid was transfected into CHO cells. The cell lines that stably expressed the CHO cells of porcine SRA or MARCO were constructed through 96 orifice cell passages and G418 drug screening. The cell lines were verified by RT-PCR and Western blot. SRA and MARCO mediated the phagocytosis of bacteria: clinical pathogenesis. Pigs were dissected and labeled from the lungs by FITC, and the labeled bacteria were interacted with the constructed stable cell lines to analyze the functions of SRA and MARCO. Results: 1. The amplification results showed that the fragments of SRA and MARCO were 1341bp and 1188bp, respectively, with the expected results, and the sequencing results and the original coding sequence. The sequence of the prokaryotic plasmid PCR showed that the fragments of SRA and MARCO were 711bp and 709bp respectively, and the size of the target fragment was the same. The sequencing results were consistent with the selected exo segment sequences, indicating that the prokaryotic expression plasmid was constructed successfully. The SRA and MARCO appeared at 32kDa and 31kD, respectively, by Western blot analysis. The strip is the same as the target fragment, indicating that the polyclonal antibody prepared in the experiment can identify rSRA-c (about 32kDa) and rMARCO-c (about 31kDa), and has a good immunogenicity. (2) the size of SRA and MARCO in the PCR results of real nuclear particles is 1341bp and 1188bp respectively, which are the same as those of the coding sequence, and the sequencing results are the same as those of the coding sequence, indicating eukaryon. The successful.RT-PCR results showed that the SRA cell line appeared the size of 1341bp, and the size of the SRA was the same size. The MARCO cell line appeared the band of 1188bp, the same as the MARCO coding sequence. The Western blot knot of the cell line protein showed that the protein sample of the SRA positive cloned cell was in 55kD. The target band appeared, and the protein sample of MARCO positive cloned cells appeared at less than 55kD, which showed that the stable cell line was constructed successfully. (3) Actinobacillus pleuropneumoniae, Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes were isolated from the lungs of the clinically infected pigs. The phagocytosis of bacteria was mediated by porcine SRA and MARCO. The analysis shows that both SRA and MARCO can effectively mediate phagocytosis of Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes, but neither can mediate phagocytosis of Actinobacillus pleuropneumoniae, indicating that porcine SRA and MARCO can mediate phagocytosis of some bacteria. This study is not only to further study the pathogenic microbes of swine SRA and MARCO. It lays a foundation for revealing the role of SRA and MARCO in the pathogenic bacteria.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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