轮状病毒外壳蛋白VP7基因在拟南芥种子中表达及免疫原性分析
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摘要
为了研究开发植物来源的轮状病毒疫苗,试验采用模式植物拟南芥作为生物反应器,将轮状病毒主要中和抗原基因VP7通过农杆菌介导法转化野生型拟南芥,获得了含有VP7的转基因拟南芥稳定株系,并分析其种子表达蛋白的免疫原性。首先根据拟南芥密码子的偏好性,设计合成优化的VP7基因,构建植物表达载体p PHAP1301-His-HRVVP7;然后通过农杆菌介导的Floral Dip法转化拟南芥,收获T0代种子后播种,喷用草铵膦筛选转基因拟南芥获得T1代种子,重复上述过程最终获得T3代拟南芥种子,通过RT-PCR、Western、ELISA法等进行分析。结果表明:VP7基因已成功整合到拟南芥的基因组并进行了转录和蛋白表达,并且VP7蛋白占总可溶性蛋白的0.31%,即每克转基因拟南芥种子中所含目的蛋白为43.3μg。以转基因拟南芥稳定株系种子中的可溶性蛋白口服免疫小鼠,可使小鼠获得免疫原性。说明通过植物作为生物反应器生产轮状病毒疫苗方法可行。
To explore the development of rotavirus vaccine in the plant,the VP7 was genetically cloned to the up stream of the His and codon-optimized in Arabidopsis thaliana. p PHAP1301-His-HRVVP7 expression vecter was constructed and transformed into Arabidpsis thaliana.The VP7 gene was analyzed by RT-PCR,Western and ELISA. Furthermore,the protective response of plant derived VP7 was evaluated in mice by oral immunizations. The results revealed that expression vector of the p PHAP1301-His-HRVVP7 was successfully constructed,and transformed into Arabidpsis thaliana. Molecular analysis revealed that VP7 was efficiently expressed in plant seeds where the VP7 protein accounted for 0. 31% in the sample of TSP in Arabidopsis thaliana carrying VP7 gene. A high level VP7( 43. 3 μg) protein was expressed in each gram of VP7 transgenic seed. The immunized mice with the TSP of transformed seeds could elicit serum Ig G and mucosal Ig A aganinst VP7 protein. These data indicate that the VP7 protein produced in Arabidopsis thaliana is a potential candidate for subunit vaccine against rotavirus.
To explore the development of rotavirus vaccine in the plant,the VP7 was genetically cloned to the up stream of the His and codon-optimized in Arabidopsis thaliana. p PHAP1301-His-HRVVP7 expression vecter was constructed and transformed into Arabidpsis thaliana.The VP7 gene was analyzed by RT-PCR,Western and ELISA. Furthermore,the protective response of plant derived VP7 was evaluated in mice by oral immunizations. The results revealed that expression vector of the p PHAP1301-His-HRVVP7 was successfully constructed,and transformed into Arabidpsis thaliana. Molecular analysis revealed that VP7 was efficiently expressed in plant seeds where the VP7 protein accounted for 0. 31% in the sample of TSP in Arabidopsis thaliana carrying VP7 gene. A high level VP7( 43. 3 μg) protein was expressed in each gram of VP7 transgenic seed. The immunized mice with the TSP of transformed seeds could elicit serum Ig G and mucosal Ig A aganinst VP7 protein. These data indicate that the VP7 protein produced in Arabidopsis thaliana is a potential candidate for subunit vaccine against rotavirus.
引文
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[13]何湘君,钱渊,靖宇,等.GI型A组轮状病毒地方株VP7基因在杆状病毒系统中的表达[J].病毒学报,1999,15(1):75-77.
[14]CHOI N W,ESTES M K,LANGRIDGE W H.Synthesis of a ricin toxin B subunit-rotavirus vp7 fusion protein in potato[J].Mol Biotechnol,2006,32(2):117-128.
[15]WEIGEL D,GLAZEBROOK J.Arabidopsis:A laboratory manual[M].Cold Spring Harbor,N.Y.:Cold Spring Harbor Laboratory Press,c2002.
[16]MASON H S,LAM D M,ARNTZEN C J.Expression of hepatitis B surface antigen in transgenic plants[J].Proc Natl Acad Sci USA,1992,89(24):11745-11749.
[17]HAQ T A,MASON H S,CLEMENT J D,et al.Oral immunization with a recombinant bacterial antigen produced in transgenic plants[J].Science,1995,268(5211):714-716.
[2]ARNTZEN C J.High-tech herbal medicine:plant-based vaccines[J].Nat Biotechnol,1997,15(3):221-222.
[3]DONG J L,LIANG B G,JIN Y S,et al.Oral immunization with p Bs VP6-transgenic alfalfa protects mice against rotavirus infection[J].Virology,2005,339(2):153-163.
[4]ZHOU B,ZHANG Y,WANG X,et al.Oral administration of plantbased rotavirus VP6 induces antigen-specific Ig As,Ig Gs and passive protection in mice[J].Vaccine,2010,28(37):6021-6027.
[5]TACKET C O,MASON H S,LONSONSKY G,et al.Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato[J].Nat Med,1998,4(5):607-609.
[6]ARAKAWA T,YU J,LANGRIDGE W H R.Synthesis of a cholera toxin B subunit-rotavirus NSP4 fusion protein in potato[J].Plant Cell Reports,2001,20(3):343-348.
[7]YU J,LANGRIDGE W H.A plant-based multicomponent vaccine protects mice from enteric diseases[J].Nat Biotechnol,2001,19(6):548-552.
[8]KIM T G,LANGRIDGE W H.Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato[J].Plant Cell Rep,2003,21(9):884-890.
[9]THAGUN C,SRISALA J,SRITUNYALUCKSANA K,et al.Arabidopsis-derived shrimp viral-binding protein,PmRab7 can protect white spot syndrome virus infection in shrimp[J].J Biotechnol,2012,161(1):60-67.
[10]PARASHAR U D,GIBSON C J,BRESSE J S,et al.Rotavirus and severe childhood diarrhea[J].Emerg Infect Dis,2006,12(2):304-306.
[11]PARASHAR U D,BRESEE J S,GLASS R I.The global burden of diarrhoeal disease in children[J].Bull World Health Organ,2003,81(4):236.
[12]袁力勇,刘勇,李春宏,等.轮状病毒VP7基因在大肠杆菌中的表达及其免疫原性[J].生物工程学报,2001,17(2):145-149.
[13]何湘君,钱渊,靖宇,等.GI型A组轮状病毒地方株VP7基因在杆状病毒系统中的表达[J].病毒学报,1999,15(1):75-77.
[14]CHOI N W,ESTES M K,LANGRIDGE W H.Synthesis of a ricin toxin B subunit-rotavirus vp7 fusion protein in potato[J].Mol Biotechnol,2006,32(2):117-128.
[15]WEIGEL D,GLAZEBROOK J.Arabidopsis:A laboratory manual[M].Cold Spring Harbor,N.Y.:Cold Spring Harbor Laboratory Press,c2002.
[16]MASON H S,LAM D M,ARNTZEN C J.Expression of hepatitis B surface antigen in transgenic plants[J].Proc Natl Acad Sci USA,1992,89(24):11745-11749.
[17]HAQ T A,MASON H S,CLEMENT J D,et al.Oral immunization with a recombinant bacterial antigen produced in transgenic plants[J].Science,1995,268(5211):714-716.