本文選題：GGTA1基因 + 小型豬��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬是人異種器官移植的理想供體,由α-1,3半乳糖苷(α-1,3Gal)引起的豬到人器官移植的超急性排斥反應(yīng)已經(jīng)基本解決,但是隨之而來的細(xì)胞性排斥反應(yīng)以及血栓和炎癥成為目前亟待解決的問題,豬白細(xì)胞抗原(swine leukocyte antigen,SLA)是引起異種移植細(xì)胞性免疫排斥的主要因素之一,轉(zhuǎn)人內(nèi)皮蛋白C受體(endothelial cell protein C receptor,EPCR)可以有效降低豬到人器官移植引起的血栓和炎癥。本研究主要探討了α-1,3半乳糖苷轉(zhuǎn)移酶(α-1,3-galactosyltransferase,GGTA1)基因敲除巴馬小型豬(Bama minipig,BMP)的健康水平以及SLA等位基因和單倍型為異種器官移植研究篩選細(xì)胞排斥低的供體,制備可以降低異種器官移植引起的炎癥和血栓反應(yīng)的轉(zhuǎn)hEPCR基因豬。本論文共有三部分研究內(nèi)容,分別為:1.GGTA1基因敲除巴馬小型豬的繁育及健康水平檢測。本課題組制備的GGTA1基因敲除(α-1,3-galactosyltransferase knockout,GTKO)巴馬小型豬已經(jīng)繁育到F3代。本研究跟蹤了GTKO巴馬小型豬的遺傳、窩產(chǎn)仔數(shù)和血常規(guī)血生化指標(biāo),評估其繁殖和健康狀況。通過PCR產(chǎn)物測序鑒定仔豬GGTA1基因敲除類型,FITC-GSIB4與仔豬外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMC)孵育后流式細(xì)胞術(shù)檢測α-1,3-Gal表達(dá),以窩產(chǎn)仔數(shù)測定繁殖力,以血常規(guī)和血生化檢測反映健康狀況。結(jié)果顯示,GGTA1基因的遺傳符合孟德爾分離定律;GGTA1敲除純合子(GGTA1-/-)仔豬的流式檢測熒光強(qiáng)度低;GTKO巴馬豬母豬頭胎窩產(chǎn)仔數(shù)為7.38±1.64頭,經(jīng)產(chǎn)母豬窩產(chǎn)仔數(shù)為9.43±1.68頭,與已有報(bào)道的普通巴馬小型豬繁殖力無明顯差異;血常規(guī)血生化檢測的各項(xiàng)指標(biāo)與普通巴馬豬基本無差異�？傊�,連續(xù)3代GTKO巴馬仔豬遺傳穩(wěn)定,繁殖力正常,生理健康。GTKO巴馬小型豬可作為異種器官移植研究的可靠供體。2.GGTA1基因敲除巴馬小型豬SLA單倍型分型。本研究主要對SLA基因上5個(gè)位點(diǎn)(SLA-1、SLA-2、SLA-3、SLA-DRB1和SLA-DQB1)進(jìn)行研究。采用轉(zhuǎn)錄本PCR產(chǎn)物直接測序的方法獲得GTKO巴馬小型豬SLA等位基因和單倍型,補(bǔ)體依賴的淋巴細(xì)胞毒試驗(yàn)(Complementdependent cytotoxicity,CDC)檢測不同SLA單倍型豬的PBMC與人血清反應(yīng)的細(xì)胞死亡率。結(jié)果發(fā)現(xiàn),GGTA1基因敲除巴馬小型豬群的5個(gè)SLA位點(diǎn)共發(fā)現(xiàn)15個(gè)等位基因,其中SLA-2*05:07(序列號:KX022946)和SLA-3*03:10(序列號:KX022947)2條序列為新發(fā)現(xiàn)序列,GGTA1基因敲除巴馬小型豬群共有5個(gè)SLA單倍型,分別為Hp-80.27、Hp-81.27、Hp-83a.45、Hp-82.44和Hp-83b.10c。4種雜合單倍型(Hp-80.27/81.27,Hp-82.44/83b.10c,Hp-80.27/83b.10c和Hp-83a.45/83b.10c)豬CDC結(jié)果細(xì)胞死亡率都小于27.5%,Hp-80.27/81.27和Hp-82.44/83b.10c單倍型的豬CDC結(jié)果細(xì)胞死亡率都小于17%。結(jié)果表明,不同SLA單倍型豬PBMC與人血清CDC試驗(yàn)結(jié)果不同,Hp-80.27/81.27和Hp-82.44/83b.10c單倍型的豬CDC結(jié)果細(xì)胞死亡率更低,更適合做異種器官移植研究供體。3.轉(zhuǎn)hEPCR基因豬的制備。本研究構(gòu)建廣泛性表達(dá)啟動(dòng)子CAG調(diào)控的hEPCR基因表達(dá)載體pCAGGS-hEPCR-Puro,轉(zhuǎn)染巴馬五指山雜交豬胎兒成纖維細(xì)胞,通過體細(xì)胞克隆技術(shù)制備轉(zhuǎn)基因克隆豬。利用PCR技術(shù)對克隆仔豬進(jìn)行轉(zhuǎn)基因鑒定,同時(shí)通過實(shí)時(shí)熒光定量PCR(qRT-PCR)、Western Blot及免疫組化方法分析hEPCR在轉(zhuǎn)基因豬的各個(gè)器官中的表達(dá)。本研究成功構(gòu)建了pCAGGS-hEPCR-Puro載體,轉(zhuǎn)染細(xì)胞后篩選出71個(gè)有效克隆點(diǎn),共得到2頭轉(zhuǎn)hEPCR陽性仔豬,qRT-PCR檢測發(fā)現(xiàn)hEPCR基因在克隆仔豬肝臟、腎臟、心臟、脾臟、胰臟、肺臟、主動(dòng)脈等主要器官都有表達(dá),在主動(dòng)脈、胰臟、肺臟中的表達(dá)水平較高。Western Blot和免疫組化結(jié)果表明hEPCR蛋白在耳朵、主動(dòng)脈,胰臟、心臟和腎臟都有表達(dá)。本研究成功制備了轉(zhuǎn)hEPCR基因豬,并且hEPCR基因在克隆豬的主要器官組織都有較高的表達(dá)水平,為異種器官移植研究制備了良好供體。
[Abstract]:Porcine is an ideal donor for xenotransplantation. The hyperacute rejection of porcine to human organ transplantation caused by alpha -1,3 galactoside (alpha -1,3Gal) has been basically solved, but the attendant cellular rejection and thrombus and inflammation are the problems to be solved urgently. The swine leukocyte antigen (SLA) is the leading factor. One of the main factors for xenograft rejection is that the endothelial cell protein C receptor (EPCR) C receptor (EPCR) can effectively reduce the thrombosis and inflammation caused by human organ transplantation. This study mainly discusses the alpha galactosidase (alpha -1,3-galactosyltransferase, GGTA1) gene knockout in Bama. The health level of Bama Minipig (BMP) and the SLA allele and haplotype for xenotransplantation screening the low cell rejection donor for the preparation of hEPCR transgenic pigs that can reduce the inflammatory and thrombus reaction caused by xenotransplantation. This paper has three parts: 1.GGTA1 gene knockout Bama miniature pig GGTA1 gene knockout (alpha -1,3-galactosyltransferase knockout, GTKO) Bama miniature pigs have been bred to the F3 generation. This study traced the inheritance, litter size and blood biochemical indexes of GTKO Bama miniature pigs, and evaluated their reproductive and health status. The offspring were sequenced and identified by PCR products. GGTA1 gene knockout type, FITC-GSIB4 and peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) of piglets were incubated by flow cytometry to detect the expression of alpha -1,3-Gal. The fecundity was measured by litter size, and the health status was reflected by blood routine and blood biochemical tests. The results showed that the inheritance of GGTA1 gene conforms to the Mendel law of separation. The flow detection fluorescence intensity of GGTA1 knockout homozygote (GGTA1-/-) piglets was low, the number of litter size in the head litter of GTKO Bama sows was 7.38 + 1.64, the number of litter size of the sows was 9.43 + 1.68 heads, and there was no significant difference from the common Bama miniature pig's fecundity, and the indexes of blood routine blood biochemistry were basically no different from that of normal Bama pigs. In a word, the 3 generations of GTKO Bama piglets have been genetically stable, and the reproductive capacity is normal. The physiological healthy.GTKO Bama miniature pig can be used as a reliable donor.2.GGTA1 gene for SLA haplotyping of the Bama miniature pig. This study mainly studies the 5 loci of the SLA gene (SLA-1, SLA-2, SLA-3, SLA-DRB1 and SLA-DQB1). The GTKO Bama miniature pig SLA allele and haplotype were obtained by direct sequencing of the PCR product, and the complement dependent lymphocyte toxicity test (Complementdependent cytotoxicity, CDC) was used to detect the cell death rate of PBMC and human serum reaction in different SLA haplotype pigs. The results showed that the GGTA1 gene knocked out 5 SLA loci of the Bama miniature pig. 15 alleles were found, including 2 sequences of SLA-2*05:07 (sequence number: KX022946) and SLA-3*03:10 (sequence number: KX022947). There were 5 SLA haplotypes of GGTA1 gene knockout Bama miniature pigs, Hp-80.27, Hp-81.27, Hp-83a.45, Hp-82.44 and Hp-83b.10c.4 hybrid haplotypes. 80.27/83b.10c and Hp-83a.45/83b.10c) the mortality of pig CDC cells was less than 27.5%. The results of CDC results in Hp-80.27/81.27 and Hp-82.44/83b.10c haplotype pigs were less than that of 17%., and the results of PBMC in different SLA haplotype pigs were different from those of CDC in human serum, and the result cells died in Hp-80.27/81.27 and Hp-82.44/83b.10c haplotypes. The death rate is lower, and it is more suitable for the preparation of the donor.3. transgenic hEPCR gene pig. This study constructs a hEPCR gene expression vector pCAGGS-hEPCR-Puro regulated by the promoter CAG, which is widely used to transfect the fetal fibroblasts of Bama Five Fingers Group hybrid pig, and the transgenic cloned pig is prepared by somatic cell clon technology. The PCR technology is used. The cloning of piglets was genetically modified, and the expression of hEPCR in various organs of transgenic pigs was analyzed by real-time quantitative PCR (qRT-PCR), Western Blot and immunohistochemical method. The pCAGGS-hEPCR-Puro vector was successfully constructed. After transfecting the cells, 71 effective clones were screened and 2 hEPCR positive piglets were obtained, qRT-PCR was obtained. The expression of hEPCR gene in the liver, kidney, kidney, heart, spleen, pancreas, pancreas, lung and aorta of piglets was expressed. The high expression level of.Western Blot and immunohistochemistry in aorta, pancreas and lung showed that hEPCR protein was expressed in the ear, active vein, pancreas, heart and kidney. Transgenic hEPCR pigs and hEPCR genes have higher expression level in the main organs and tissues of the cloned pigs, and have prepared good donors for xenotransplantation.

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R617;Q78

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本文編號：1777840