广西巴马小型猪α-1,3-半乳糖转移酶基因RNAi载体构建与检测
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摘要
α-1,3-半乳糖转移酶基因(α-1,3 GT)在异种器官移植中的免疫排斥反应有重要的作用,本研究旨在构建α-1,3-半乳糖转移酶基因的干扰载体,在m RNA和蛋白质水平鉴定并筛选出干扰效果最佳的序列。根据巴马小型猪GGTA1基因CDS序列,设计合成3对特异性单链si RNA序列以及一对阴性序列,构建GGTA1 RNA干扰载体分别命名为p LLU2G-sh GGTA1-1、p LLU2G-sh GGTA1-2、p LLU2G-sh GGTA1-3和p LLU2G-sh GGTA1-NC。将构建好的载体分别转染转染PK15细胞,通过q RT-PCR和Western blot检测GGTA1 m RNA及其蛋白的相对表达量。结果显示本研究成功构建了3个重组RNAi表达载体,与转染p LLU2G-NC和空白组相比,p LLU2G-sh GGTA1-1、p LLU2G-sh GGTA1-2和p LLU2G-sh GGTA1-3转染组的GGTA1 m RNA和蛋白表达量均明显下降,对m RNA表达的抑制效率分别为85.02%、86.05%和83.85%。本研究成功获得有效抑制广西巴马小型猪GGTA1基因表达的RNAi载体,为下一步生产沉默GGTA1基因广西巴马小型猪奠定基础。
α-1,3 galactose transferase gene play an important role in hyperacute immune rejection of clinical Xenotransplantation. The objective of this study was to construct the RNAi vectors of porcine α-1,3-galactose transferase gene and screen the best RNAi vector on cell m RNA and protein level. According to CDS sequence of Guangxi Bama mini-pig's α-1,3-galactose transferase gene cloned in our lab, three si RNA sequences and a negative sequence were designed and synthesized, directly cloned into p LLU2 G vector to produce p LLU2G-sh GGTA1-1,p LLU2G-sh GGTA1-2, p LLU2G-sh GGTA1-3 and p LLU2G-sh GGTA1-NC vectors. The constructed vectors were transfected into PK-15 cells, relative transcription level of GGTA1 gene m RNA and protein expression were determined by Real-time PCR and Western blot, respectively. The results showed that 3 GGTA1 gene RNAi vectors were constructed successfully. Compared with p LLU2G-sh GGTA1-NC, the relative level of GGTA1 gene m RNA and protein expression decreased in PK-15 cells which transfected with p LLU2G-sh GGTA1-1,p LLU2G-sh GGTA1-2 and p LLU2G-sh GGTA1-3. The m RNA inhibition ratios were 85.02%, 86.05% and 83.85,respectively. In conclusion, effective RNAi vectors for α-1,3-galactose transferase gene have been successfully constructed, which would lay a foundation for further producing and breeding GGTA1 gene silencing Guangxi Bama mini-pig.
α-1,3 galactose transferase gene play an important role in hyperacute immune rejection of clinical Xenotransplantation. The objective of this study was to construct the RNAi vectors of porcine α-1,3-galactose transferase gene and screen the best RNAi vector on cell m RNA and protein level. According to CDS sequence of Guangxi Bama mini-pig's α-1,3-galactose transferase gene cloned in our lab, three si RNA sequences and a negative sequence were designed and synthesized, directly cloned into p LLU2 G vector to produce p LLU2G-sh GGTA1-1,p LLU2G-sh GGTA1-2, p LLU2G-sh GGTA1-3 and p LLU2G-sh GGTA1-NC vectors. The constructed vectors were transfected into PK-15 cells, relative transcription level of GGTA1 gene m RNA and protein expression were determined by Real-time PCR and Western blot, respectively. The results showed that 3 GGTA1 gene RNAi vectors were constructed successfully. Compared with p LLU2G-sh GGTA1-NC, the relative level of GGTA1 gene m RNA and protein expression decreased in PK-15 cells which transfected with p LLU2G-sh GGTA1-1,p LLU2G-sh GGTA1-2 and p LLU2G-sh GGTA1-3. The m RNA inhibition ratios were 85.02%, 86.05% and 83.85,respectively. In conclusion, effective RNAi vectors for α-1,3-galactose transferase gene have been successfully constructed, which would lay a foundation for further producing and breeding GGTA1 gene silencing Guangxi Bama mini-pig.
引文
Bao Y.H.,Guo Y.C.,Zhao Z.H.,and Li N.,2010,The application prospect of RNAi technology in animal breeding for disease resistance,Zhongguo Shouyi Xuebao(Chinese Journal of Veterinary Science),(10):1402-1408(鲍永华,郭永臣,赵志辉,李宁,2010,RNA干扰技术在动物抗病育种中的应用前景,中国兽医学报,(10):1402-1408)
Brummelkamp T.R.,Bernards R.,and Agami R.,2002,A system for stable expression of short interfering RNAs in mammalian cells,Science,296(5567):550-553
Bühler L.,Friedman T.,Iacomini J.,and Cooper D.K.C.,1999,Xenotransplantation-state of the art-update,Front Biosci.4:D416-D432
Caplen N.J.,Parrish S.,Imani F.,Fire A.,and Morgan R.A.,2001,Specific inhibition of gene expression by small double-stranded RN As in invertebrate and vertebrate systems,Proc.Natl.Acad.Sci.USA,98(17):9742-9747
Chen S.J.,2012,Construction and detection of RNA interference Vectors for silencing over-expression vector and RNAi expression vector ofα-1,3-galactosyltransferase gene from guangxi bama-mini pig,Thesis for M.S.,Guangxi University,Supervisor:Guo Y.F.,pp.1-60(陈时锦,2012,广西巴马小型猪α-1,3-半乳糖转移酶基因过表达载体和RNAi载体的构建及其沉默效率检测,硕士学位论文,广西大学,导师:郭亚芬,pp.1-60)
Chen S.P.,Zhou H.T.,Cai J.H.,Wang F.L.,Xu M.X.,and Fu S.D.,2014,Influence of tandem gene survivin and CDK1 sh RNAs expression vector on nasopharyngeal carcinoma cell line CNE-2 growth,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(2):239-247(陈淑萍,周红桃,蔡俊宏,王福利,许茂轩,符生苗,2014,联合基因Survivin、CDK1的sh RNAs靶向干扰对鼻咽癌CNE-2细胞增值的影响,基因组学与应用生物学,33(2):239-247)
Cooper D.K.,Good A.H.,Koren E.,Oriol R.,Malcolm A.J.,Ippolito R.M.,Neethling F.A.,Ye Y.,Romano E.,and Zuhdi N.,1993,Identification of alpha-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies:relevance to discordant xenografting in man,Transpl.Immunol.,1(3):198-205
Dai Y.,Vaught T.D.,Boone J.,Chen S.H.,Phelps C.J.,Ball S.,Monahan J.A.,Jobst P.M.,Mc Creath K.J.,Lamborn A.E.,Cowell-Lucero J.L.,Wells K.D.,Colman A.,Polejaeva I.A.,and Ayares D.L.,2002.Targeted disruption of theα-1,3-galactosyltransferase gene in cloned pigs,Nat.Biotechnol.,20(3):251-255
Gu Y.C.,2008,The research on inhibitory effects of silencingα-1,3-GT expression by lentvirus-mediated,Thesis for M.S.,Tianjing Yike Daxue Waikexue(Puwai),Supervisor:Zhang Z.X.,pp.1-50(谷雅川,2008,慢病毒介导的RNAi抑制小鼠血管内皮细胞α-1,3-GT表达的研究,硕士学位论文,天津医科大学外科学(普外),导师:章志翔,pp.1-50)
Lai L.,Kolber-Simonds D.,Park K.W.,Cheong H.T.,Greenstein J.L.,Im G.S.,Melissa S.,Bonk A.,Rieke A.,Day B.N.,Murphy C.N.,Carter D.B.,Hawley R.J.,and Prather R.S.,2002,Production ofα-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning,Science,295(5557):1089-1092
Liu M.,Zhu S.M.,Zheng H.,Wang Y.,Wang Z.,Yang Y.J.,Wu Y.X.,Zeng Y.Z.,and Wang Y.P.,2012,Cloning of splicing variants ofα-1,3-galactosyltransferase c DNA of Chinese Banna mini pig inbred line and it expression in human cells,Sichuan Daxue Xuebao(Journal of Sichuan University),(02):145 -150(刘美,朱圣明,郑鸿,王宇,王竹,杨娅君,伍艳霞,曾养志,王艳萍,2012,中国近交系版纳微型猪α-1,3-半乳糖基转移酶基因剪接变异体的克隆及其在人类细胞中的表达,四川大学学报(医学版),(02):145-150)
Niu J.H.,Guo Y.D.,Jian H.,2009,Progress in the study of RNAi mediated plant root-knot nematodes resistant genetic engineering,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),28(1):167-173(牛俊海,郭仰东,简恒,RNA干扰在植物抗根结线虫病基因工程应用中的研究进展,基因组学与应用生物学,28(1):167-173)
Posekany K.J.,Pittman H.K.,Bradfield J.F.,Haisch C.E.,and Verbanac K.M.,2002,Induction of cytolytic anti-Gal antibodies inα-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria,Infect Immun.,70(11):6215-6222
Qin F.,Zhu S.M.,Zheng H.,Wang Z.,Wang Y.,Zuo Y.,Chen J.,Sun W.L.,and Wang Y.P.,2010,Establishment and identification of human lung adenocarcinoma cell line stably expressing theα-1,3-galactosyltransferase gene from pig,Sichuan Daxue Xuebao(Journal of Sichuan University),(02):
194 -198(秦凤,朱圣明,郑鸿,王竹,王宇,左莹,陈娟,孙蔚亮,王艳萍,2010,稳定表达猪α-1,3-半乳糖苷基转移酶基因的人肺腺癌细胞系的建立和鉴定,四川大学学报(医学版),(02):194-198)
Sun Q.Y.,Chen G.,Guo H.,Chen S.,Wang W.Y.,and Xiong Y.L.,2003,Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor,Toxicon,42(3):257-262.
Timmons L.,Court D.L.,and Fire A.,2001,Ingestion of bacterially expressed ds RNAs can produce specific and potent genetic interference in Caenorhabditis elegans,Gene,263(1-2):103-112
Wang A.D.,Lan G.Q.,and Guo Y.F.,2010,Genetic breeding of Guangxi Bama mini-pig,Shiyan Dongwu Kexue(Laboratory Animal Science),(01):60-63(王爱德,兰干球,郭亚芬,2010,广西巴马小型猪的培育,实验动物科学,(01):60-63)
Xie Q.,Jiang J.R.,Wang D.,Shi D.S.,Xie T.S.,Chen S.B.,and Liu Q.Y.,2011,Construction and analysis of buffalo inhibinα-subunit gene RNAi vectors,Zhongguo Shouyi Xuebao(Chinese Journal of Veterinary Science),(7):1070-1075(谢琴,蒋建荣,王丹,石德顺,谢体三,陈施蓓,刘庆友,2011,水牛抑制素α基因RNAi载体构建与检测,中国兽医学报,(7):1070-1075)
Yan Q.J.,Zhang Y.F.,Yang J.,Ding Q.,Wang J.G.,Jiang H.W.,Zhao H.,Xu K.,Gong J.,Li L.J.,Liu H.J.,Guo J.L.,and Liu E.J.,2004,The porcineα-1,3-galactosyltransferase gene si RNA targeted heterozygous hepatocyte negative express GT,Zhonghua Ganzangbing Zazhi(Chinese Journal of Hepatology),12(8):482-484(严泉剑,张元芳,杨捷,丁强,王继功,姜昊文,赵鸿,徐可,龚健,李六金,刘海军,郭金龙,刘恩靖,2004,α-1,3-GT si RNA打靶杂合子猪肝细胞阴性表达半乳糖转,移酶,中华肝脏病杂志,12(8):482-484)
Zhu M.,Wang S.S.,and Xia Z.X.,2005,Inhibition of xenogeneic response in porcine endothelium using RNA interference,Transplantation,79(3):289-296
Zhu S.M.,Zheng H.,Qin F.,Wang Y.,Wang Z.,Luo Z.G.,Ding J.,and Wang Y.P.,2009,Construction of xenoantigenα-1,3-GT gene expression vector regulated by h TERT promoter and its targeting expression in human lung cancer cell,Zhongguo Zhongliu Shengwu Zhiliao Zazhi(Chinese Journal of Cancer Biotherapy)(05):452-457(朱圣明,郑鸿,秦凤,王宇,王竹,骆志国,丁珺,王艳萍,2009,h TERT启动子调控的异种抗原基因α-1,3-GT表达载体的构建及其在肺癌细胞中的靶向表达,中国肿瘤生物治疗杂志,(05):452-457)
Zhu X.X.,Quan S.N.,Huang Y.,and Lu S.S.,2013,The application of somatic cell nuclear transfer technology on porcine genetic modification,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(6):692-699(朱向星,全守能,黄勇,卢晟盛,2013,体细胞核移植技术在猪基因修饰中的应用,基因组学与应用生物学,33(6):692-699)
Brummelkamp T.R.,Bernards R.,and Agami R.,2002,A system for stable expression of short interfering RNAs in mammalian cells,Science,296(5567):550-553
Bühler L.,Friedman T.,Iacomini J.,and Cooper D.K.C.,1999,Xenotransplantation-state of the art-update,Front Biosci.4:D416-D432
Caplen N.J.,Parrish S.,Imani F.,Fire A.,and Morgan R.A.,2001,Specific inhibition of gene expression by small double-stranded RN As in invertebrate and vertebrate systems,Proc.Natl.Acad.Sci.USA,98(17):9742-9747
Chen S.J.,2012,Construction and detection of RNA interference Vectors for silencing over-expression vector and RNAi expression vector ofα-1,3-galactosyltransferase gene from guangxi bama-mini pig,Thesis for M.S.,Guangxi University,Supervisor:Guo Y.F.,pp.1-60(陈时锦,2012,广西巴马小型猪α-1,3-半乳糖转移酶基因过表达载体和RNAi载体的构建及其沉默效率检测,硕士学位论文,广西大学,导师:郭亚芬,pp.1-60)
Chen S.P.,Zhou H.T.,Cai J.H.,Wang F.L.,Xu M.X.,and Fu S.D.,2014,Influence of tandem gene survivin and CDK1 sh RNAs expression vector on nasopharyngeal carcinoma cell line CNE-2 growth,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(2):239-247(陈淑萍,周红桃,蔡俊宏,王福利,许茂轩,符生苗,2014,联合基因Survivin、CDK1的sh RNAs靶向干扰对鼻咽癌CNE-2细胞增值的影响,基因组学与应用生物学,33(2):239-247)
Cooper D.K.,Good A.H.,Koren E.,Oriol R.,Malcolm A.J.,Ippolito R.M.,Neethling F.A.,Ye Y.,Romano E.,and Zuhdi N.,1993,Identification of alpha-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies:relevance to discordant xenografting in man,Transpl.Immunol.,1(3):198-205
Dai Y.,Vaught T.D.,Boone J.,Chen S.H.,Phelps C.J.,Ball S.,Monahan J.A.,Jobst P.M.,Mc Creath K.J.,Lamborn A.E.,Cowell-Lucero J.L.,Wells K.D.,Colman A.,Polejaeva I.A.,and Ayares D.L.,2002.Targeted disruption of theα-1,3-galactosyltransferase gene in cloned pigs,Nat.Biotechnol.,20(3):251-255
Gu Y.C.,2008,The research on inhibitory effects of silencingα-1,3-GT expression by lentvirus-mediated,Thesis for M.S.,Tianjing Yike Daxue Waikexue(Puwai),Supervisor:Zhang Z.X.,pp.1-50(谷雅川,2008,慢病毒介导的RNAi抑制小鼠血管内皮细胞α-1,3-GT表达的研究,硕士学位论文,天津医科大学外科学(普外),导师:章志翔,pp.1-50)
Lai L.,Kolber-Simonds D.,Park K.W.,Cheong H.T.,Greenstein J.L.,Im G.S.,Melissa S.,Bonk A.,Rieke A.,Day B.N.,Murphy C.N.,Carter D.B.,Hawley R.J.,and Prather R.S.,2002,Production ofα-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning,Science,295(5557):1089-1092
Liu M.,Zhu S.M.,Zheng H.,Wang Y.,Wang Z.,Yang Y.J.,Wu Y.X.,Zeng Y.Z.,and Wang Y.P.,2012,Cloning of splicing variants ofα-1,3-galactosyltransferase c DNA of Chinese Banna mini pig inbred line and it expression in human cells,Sichuan Daxue Xuebao(Journal of Sichuan University),(02):145 -150(刘美,朱圣明,郑鸿,王宇,王竹,杨娅君,伍艳霞,曾养志,王艳萍,2012,中国近交系版纳微型猪α-1,3-半乳糖基转移酶基因剪接变异体的克隆及其在人类细胞中的表达,四川大学学报(医学版),(02):145-150)
Niu J.H.,Guo Y.D.,Jian H.,2009,Progress in the study of RNAi mediated plant root-knot nematodes resistant genetic engineering,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),28(1):167-173(牛俊海,郭仰东,简恒,RNA干扰在植物抗根结线虫病基因工程应用中的研究进展,基因组学与应用生物学,28(1):167-173)
Posekany K.J.,Pittman H.K.,Bradfield J.F.,Haisch C.E.,and Verbanac K.M.,2002,Induction of cytolytic anti-Gal antibodies inα-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria,Infect Immun.,70(11):6215-6222
Qin F.,Zhu S.M.,Zheng H.,Wang Z.,Wang Y.,Zuo Y.,Chen J.,Sun W.L.,and Wang Y.P.,2010,Establishment and identification of human lung adenocarcinoma cell line stably expressing theα-1,3-galactosyltransferase gene from pig,Sichuan Daxue Xuebao(Journal of Sichuan University),(02):
194 -198(秦凤,朱圣明,郑鸿,王竹,王宇,左莹,陈娟,孙蔚亮,王艳萍,2010,稳定表达猪α-1,3-半乳糖苷基转移酶基因的人肺腺癌细胞系的建立和鉴定,四川大学学报(医学版),(02):194-198)
Sun Q.Y.,Chen G.,Guo H.,Chen S.,Wang W.Y.,and Xiong Y.L.,2003,Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor,Toxicon,42(3):257-262.
Timmons L.,Court D.L.,and Fire A.,2001,Ingestion of bacterially expressed ds RNAs can produce specific and potent genetic interference in Caenorhabditis elegans,Gene,263(1-2):103-112
Wang A.D.,Lan G.Q.,and Guo Y.F.,2010,Genetic breeding of Guangxi Bama mini-pig,Shiyan Dongwu Kexue(Laboratory Animal Science),(01):60-63(王爱德,兰干球,郭亚芬,2010,广西巴马小型猪的培育,实验动物科学,(01):60-63)
Xie Q.,Jiang J.R.,Wang D.,Shi D.S.,Xie T.S.,Chen S.B.,and Liu Q.Y.,2011,Construction and analysis of buffalo inhibinα-subunit gene RNAi vectors,Zhongguo Shouyi Xuebao(Chinese Journal of Veterinary Science),(7):1070-1075(谢琴,蒋建荣,王丹,石德顺,谢体三,陈施蓓,刘庆友,2011,水牛抑制素α基因RNAi载体构建与检测,中国兽医学报,(7):1070-1075)
Yan Q.J.,Zhang Y.F.,Yang J.,Ding Q.,Wang J.G.,Jiang H.W.,Zhao H.,Xu K.,Gong J.,Li L.J.,Liu H.J.,Guo J.L.,and Liu E.J.,2004,The porcineα-1,3-galactosyltransferase gene si RNA targeted heterozygous hepatocyte negative express GT,Zhonghua Ganzangbing Zazhi(Chinese Journal of Hepatology),12(8):482-484(严泉剑,张元芳,杨捷,丁强,王继功,姜昊文,赵鸿,徐可,龚健,李六金,刘海军,郭金龙,刘恩靖,2004,α-1,3-GT si RNA打靶杂合子猪肝细胞阴性表达半乳糖转,移酶,中华肝脏病杂志,12(8):482-484)
Zhu M.,Wang S.S.,and Xia Z.X.,2005,Inhibition of xenogeneic response in porcine endothelium using RNA interference,Transplantation,79(3):289-296
Zhu S.M.,Zheng H.,Qin F.,Wang Y.,Wang Z.,Luo Z.G.,Ding J.,and Wang Y.P.,2009,Construction of xenoantigenα-1,3-GT gene expression vector regulated by h TERT promoter and its targeting expression in human lung cancer cell,Zhongguo Zhongliu Shengwu Zhiliao Zazhi(Chinese Journal of Cancer Biotherapy)(05):452-457(朱圣明,郑鸿,秦凤,王宇,王竹,骆志国,丁珺,王艳萍,2009,h TERT启动子调控的异种抗原基因α-1,3-GT表达载体的构建及其在肺癌细胞中的靶向表达,中国肿瘤生物治疗杂志,(05):452-457)
Zhu X.X.,Quan S.N.,Huang Y.,and Lu S.S.,2013,The application of somatic cell nuclear transfer technology on porcine genetic modification,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(6):692-699(朱向星,全守能,黄勇,卢晟盛,2013,体细胞核移植技术在猪基因修饰中的应用,基因组学与应用生物学,33(6):692-699)