本文選題：清道夫受體 + 原核表達(dá)��； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：清道夫受體,作為一類先天性免疫受體,在宿主先天性防御中起著重要的作用。然而,到目前為止,對(duì)豬清道夫受體的研究還非常有限。目的:為了深入了解豬清道夫受體SRA和MARCO的生物學(xué)特性,揭示SRA和MARCO在免疫中的作用,本文以豬A類清道夫受體SRA和MARCO為研究對(duì)象,利用原核表達(dá)系統(tǒng)對(duì)受體的胞外區(qū)域進(jìn)行表達(dá),制備純化的重組蛋白,以重組蛋白為免疫原,制備多抗血清,驗(yàn)證多抗血清的特異性。此外,從臨床發(fā)病豬的肺部分離細(xì)菌,構(gòu)建豬SRA或MARCO的穩(wěn)定細(xì)胞系,檢測(cè)SRA和MARCO介導(dǎo)對(duì)細(xì)菌的吞噬功能,揭示其生物學(xué)功能。材料與方法:①豬SRA和MARCO的分子克隆、表達(dá)、純化及其抗體的制備:根據(jù)GenBank SRA(NM_001243874)和MARCO(JQ25702.1)的全基因序列,設(shè)計(jì)引物,利用豬肺組織擴(kuò)增SRA和MARCO編碼序列片段,克隆至載體pMD18-T中,測(cè)序驗(yàn)證擴(kuò)增序列的正確性。選取SRA胞外區(qū)基因片段(711bp)和MARCO胞外區(qū)基因片段(709bp),利用PCR技術(shù)獲得目的片段,分別克隆入原核表達(dá)載體pET-28a中,構(gòu)建重組原核表達(dá)載體pET-SRA-c和pET-MARCO-c。利用原核表達(dá)系統(tǒng)表達(dá)重組蛋白rSRA-c(約32kDa)和rMARCO-c(約31kDa),以親和層析技術(shù)獲得純化的rSRA-c和rMARCO-c。分別利用純化的rSRA-c和rMARCO-c為免疫原,免疫BALB/c小鼠(100ug/小鼠),制備多抗血清。②SRA和MARCO的真核表達(dá)載體及其穩(wěn)定細(xì)胞系的構(gòu)建:將編碼序列分別克隆入PCDNA3.O載體中,構(gòu)建真核表達(dá)質(zhì)粒,將真核表達(dá)質(zhì)粒轉(zhuǎn)染至CHO細(xì)胞中,通過96孔板細(xì)胞傳代及G418的藥物篩選構(gòu)建穩(wěn)定表達(dá)豬SRA或MARCO的CHO細(xì)胞的細(xì)胞系,通過RT-PCR和Western blot對(duì)細(xì)胞系進(jìn)行驗(yàn)證。?SRA和MARCO介導(dǎo)對(duì)細(xì)菌吞噬功能的分析:對(duì)臨床發(fā)病豬進(jìn)行解剖,并從肺部分離細(xì)菌并用FITC進(jìn)行標(biāo)記,將標(biāo)記好的細(xì)菌與構(gòu)建的穩(wěn)定細(xì)胞系相互作用,從而對(duì)SRA和MARCO的功能進(jìn)行分析。結(jié)果:①擴(kuò)增結(jié)果顯示SRA和MARCO的片段大小分別為1341bp和1188bp,與預(yù)期結(jié)果一致,測(cè)序結(jié)果與原編碼序列相同,表明擴(kuò)增的序列正確。原核質(zhì)粒PCR驗(yàn)證結(jié)果顯示SRA和MARCO的片段大小分別為711bp和709bp,與目的片段大小一致。測(cè)序結(jié)果與選擇的胞外段序列一致,表明原核表達(dá)質(zhì)粒構(gòu)建成功。經(jīng)Western blot分析,SRA和MARCO分別在32kDa和31kD左右處出現(xiàn)了條帶,與目的片段大小相同,表明實(shí)驗(yàn)中制備的多抗血清可以識(shí)別rSRA-c(約32kDa)和rMARCO-c(約31kDa),具有很好的免疫原性。②真核質(zhì)粒PCR結(jié)果中SRA和MARCO的大小分別為1341bp和1188bp,與編碼序列大小一致,測(cè)序結(jié)果與編碼序列相同,表明真核表達(dá)質(zhì)粒構(gòu)建成功。RT-PCR結(jié)果顯示SRA細(xì)胞系出現(xiàn)了大小為1341bp的條帶,與SRA的編碼序列大小相同,MARCO細(xì)胞系中出現(xiàn)了片段大小為1188bp的條帶,與MARCO的編碼序列大小相同。細(xì)胞系蛋白的Western blot結(jié)果顯示SRA的陽性克隆細(xì)胞蛋白樣在55kD處出現(xiàn)了目的條帶,MARCO的陽性克隆細(xì)胞蛋白樣在小于55kD處出現(xiàn)了目的條帶,表明穩(wěn)定細(xì)胞系構(gòu)建成功。③從臨床發(fā)病豬的肺中分離獲得了胸膜肺炎放線桿菌、大腸桿菌、金黃色葡萄球菌及化膿鏈球菌。經(jīng)對(duì)豬SRA和MARCO介導(dǎo)對(duì)細(xì)菌吞噬功能的分析,表明豬SRA和MARCO都能有效地介導(dǎo)對(duì)大腸桿菌、金黃色葡萄球菌以及化膿鏈球菌的吞噬,但都不能介導(dǎo)對(duì)胸膜肺炎放線桿菌的吞噬,說明豬SRA和MARCO能夠介導(dǎo)對(duì)部分細(xì)菌的吞噬。本研究不僅為進(jìn)一步研究豬SRA和MARCO在病原微生物感染中的作用做好鋪墊,而且為揭示豬SRA和MARCO在病原菌中的作用奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828

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