狂犬病病毒G蛋白在水稻中的表达及遗传稳定性鉴定
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摘要
狂犬病病毒(Rabies virus, RV) G蛋白是唯一能激发机体产生细胞免疫,并诱导产生中和抗体的结构蛋白,也是狂犬病病毒中唯一糖基化的结构蛋白,此外还介导细胞融合、受体结合以及参与病毒的致病等。本研究将优化的狂犬病G基因(GenBank登录号:ABI53869.1)序列插入到中间载体pMP3上,成功构建中间载体pMP3-G,然后将中间载体pMP3-G和植物载体pCAMBIA1300通过双酶切、连接,成功地构建了植物表达载体pCAMBIA1300-G。挑选空白未转基因水稻(Oryza sativa)种子TP309消毒,在愈伤诱导培养基上31℃光照3 d,使其长出愈伤组织,用已经转入狂犬病G基因的根癌农杆菌(Agrobacterium tumefaciens)EHA105去侵染愈伤组织,并与愈伤组织共培养3 d,将狂犬病G基因导入水稻愈伤组织中。愈伤组织再经过潮霉素抗性筛选、分化、出芽、生根,获得313株植株,取313株植株的叶片通过十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide, CTAB)法提取叶片基因组DNA,对其进行PCR扩增,经核酸胶进行初步鉴定,有79株为阳性,经过炼苗、种植共获得50株阳性表达植株。分别随机挑选50株阳性植株的种子,切半,标记,将不含胚乳的一半研磨,用提取液提取蛋白后通过抗原检测试纸条和Western blot检测,结果表明,G蛋白在转基因水稻种子中成功表达,且具有良好的反应原性。从50株阳性表达植株中挑选出高表达、反应原性最好的4个株系,将这4个株系T1代中与检测阳性结果相对应的含有胚乳的另一半种子进行组织培养种植,在植株移栽大田前后,再次用CTAB法提取T1代植株叶片基因组DNA,并以水稻的内源基因水稻淀粉分支酶基因(rice starch branching enzyme,RBE4)为内参基因,通过相对荧光定量PCR(relative fluorescence quantitative PCR)方法筛选阳性植株中的纯合植株,通过绝对荧光定量PCR(absolute real-time PCR)计算纯合植株的拷贝数,结果显示,132株植株中35株为纯合植株。纯合植株的拷贝数分别为1、2、3、5、7、11、16。本研究为今后制备新型狂犬病亚单位疫苗奠定基础。
The Rabies virus(RV) G protein is the only structural protein that stimulates the body to produce cellular immunity and induces the production of neutralizing antibodies. In this study, the optimized rabies G gene sequence was inserted into the intermediate vector pMP3, and the intermediate vector p MP3-G was successfully constructed. Then the intermediate vector pMP3-G and the plant vector pCAMBIA1300 were double-digested and ligated, and the plant expression vector pCAMBIA1300-G was successfully constructed.The blank non-transgenic rice(Oryza sativa) seed TP309 was selected and disinfected. The callus was grown on the callus induction medium for 3 d at 31 °C, and the callus was inoculated with Agrobacterium tumefaciens EHA105 which had been transferred to the rabies G gene. The callus was co-cultured for 3 d, and the rabies G gene was introduced into rice callus. The callus was further screened, differentiated, germinated, rooted, and313 plants were obtained, and the leaves of 313 plants were extracted by hexadecyl trimethyl ammonium bromide(CTAB) method. A total of 79 strains were positively identified by leaf genomic DNA amplification PCR. A total of 50 positively expressed plants were obtained after refining and planting. The seeds of 50 positive plants were randomly selected, cut in half, labeled, and half of the endosperm-free seeds were ground.The extract was extracted from the protein and detected by antigen test strips and western blot. The results showed that G protein was in the transgenic rice seeds. Successfully expressed and has good reactogenicity.Four strains with high expression and best reactivity were selected from 50 strains, and the other half of the seeds containing the endosperm corresponding to the positive test results in the T1 generation of the 4 strains were planted and planted in the plant. Before and after planting the field, the genomic DNA of T1 plants was extracted by CTAB method, and the endogenous gene of rice(rice starch branching enzyme, RBE4) was used as an internal reference. The homozygous plants in positive plants were screened by relative fluorescence quantitative PCR. Absolute real-time PCR was used to calculate the copy number of homozygous plants. As a result, 35 out of 132 plants were homozygous plants. The copy number of the homozygous plants were 1, 2, 3,5, 7, 11, and 16, respectively. This study is dedicated to the expression of rabies G gene in rice and its genetic stability, laying the foundation for the preparation of new rabies subunit vaccines in the future.
The Rabies virus(RV) G protein is the only structural protein that stimulates the body to produce cellular immunity and induces the production of neutralizing antibodies. In this study, the optimized rabies G gene sequence was inserted into the intermediate vector pMP3, and the intermediate vector p MP3-G was successfully constructed. Then the intermediate vector pMP3-G and the plant vector pCAMBIA1300 were double-digested and ligated, and the plant expression vector pCAMBIA1300-G was successfully constructed.The blank non-transgenic rice(Oryza sativa) seed TP309 was selected and disinfected. The callus was grown on the callus induction medium for 3 d at 31 °C, and the callus was inoculated with Agrobacterium tumefaciens EHA105 which had been transferred to the rabies G gene. The callus was co-cultured for 3 d, and the rabies G gene was introduced into rice callus. The callus was further screened, differentiated, germinated, rooted, and313 plants were obtained, and the leaves of 313 plants were extracted by hexadecyl trimethyl ammonium bromide(CTAB) method. A total of 79 strains were positively identified by leaf genomic DNA amplification PCR. A total of 50 positively expressed plants were obtained after refining and planting. The seeds of 50 positive plants were randomly selected, cut in half, labeled, and half of the endosperm-free seeds were ground.The extract was extracted from the protein and detected by antigen test strips and western blot. The results showed that G protein was in the transgenic rice seeds. Successfully expressed and has good reactogenicity.Four strains with high expression and best reactivity were selected from 50 strains, and the other half of the seeds containing the endosperm corresponding to the positive test results in the T1 generation of the 4 strains were planted and planted in the plant. Before and after planting the field, the genomic DNA of T1 plants was extracted by CTAB method, and the endogenous gene of rice(rice starch branching enzyme, RBE4) was used as an internal reference. The homozygous plants in positive plants were screened by relative fluorescence quantitative PCR. Absolute real-time PCR was used to calculate the copy number of homozygous plants. As a result, 35 out of 132 plants were homozygous plants. The copy number of the homozygous plants were 1, 2, 3,5, 7, 11, and 16, respectively. This study is dedicated to the expression of rabies G gene in rice and its genetic stability, laying the foundation for the preparation of new rabies subunit vaccines in the future.
引文
陈腾,张锦霞,张静.2016.犬病病毒不同毒株糖蛋白免疫原性的比较研究[J].中国兽医科学,46(04):502-506(Chen T,Zhang J X,Zhang J,et al.2016.Comparative study on the immunogenicity of different Rabies virus glycoproteins[J].Chinese Veterinary Science,46(04)502-506.)
冯烨.2015.中国动物狂犬病流行病学研究[D].博士学位论文,中国人民解放军军事医学科学院,导师:涂长春pp.94.(Feng Y.2015.Epidemiology of animal rabies in China[D].Doctoral thesis.Academy of Military Medical Sciences,Chinese People's Liberation Army),Instructor:Tu C C,pp.94.)
李冰,顾劲乔.2017.中国狂犬病年会综述[J].中国工作犬业,(5):58-60.(Li B,Gu J Q.2017.Overview of 2017Rabies conference in china[J].Chinese Working Dog Industry,(5):58-60.)
孙喆,刘营,张明辉,等.2015.转OsDREB3基因大豆外源基因拷贝数的确定及标准分子的构建[J].甘肃农业大学学报,50(03):61-67.(Sun Z,Liu Y,Zhang M H,et al2015.Determination of the foreign gene copy number of OsDREB3 gene and the construction of standard molecules[J].Journal of Gansu Agricultural University,50(03):61-67.)
许倩茹,张二芹,郭军庆,等.2017.新城疫病毒F蛋白在水稻中的表达及检测[J].畜牧兽医学报,(6):1167-1172(Xu Q R,Zhang E Q,Guo J Q,et al.2017.Expression and detection of Newcastle disease virus F protein in rice[J].Journal of Animal Husbandry and Veterinary Medicine,(6):1167-1172.)
余笑波,崔海峰,俞晓平,等.2012.水稻淀粉分支酶基因(RBE4)作为转基因水稻基体标准物质的内标准基因的研究[J].农业生物技术学报,20(11):1234-1243.(Yu X B,Cui H F,Yu X P,et al.2012.Studies on rice starch branching enzyme(RBE4)as endogenous reference gene for the matrix reference material of transgenic rice(Oryza sativa L.)[J].Journal of Agricultural Biotechnology,20(11):1234-1243.)
张守峰,刘晔,赵敬慧,等.2012.从流行毒株的系统发生看中国目前狂犬病流行的主要特征[J].中国动物传染病学报,20(4):19-23.(Zhang S F,Liu Y,Zhao J H,et al.Epidemiological characterization of rabies virus isolates in china by phylogenetic analysis[J].Chinese Journal of Animal Infectious Diseases,20(4):19-23.)
Boothe J,Nykiforuk C,Shen Y,et al.2010.Seed-based expression systems for plant molecular farming[J].Plant Biotechnology Journal,8(5):588-606.
Delagneau J F,Perrin P,Atanasiu P.1981.Structure of the rabies virus:Spatial relationships of the proteins G,M1,M2 and N[J].Ann Inst Pasteur Virol,132(4):473-493.
Jiao W Q,Yin X P,Li Z Y,et al.2011.Molecular characterization of China rabies virus vaccine strain[J].Virology Journal,17(8):521-531.
Paul M,Ma K C.2011.Plant-made pharmaceuticals:Leading products and production platforms[J].Biotechnology&Applied Biochemistry,58(1):58-67.
Qiu J R,Xu Y,Yu F G.2011.Determining the copy number of exogenous gene in transgenic plant by SYBR green realtime quantitative PCR[J].Agricultural Science&Technology,12(6):829-831,835.
Rojas-Anaya E,Loza-Rubio E,Olivera-Flores M T,et al.2009.Expression of rabies virus G protein in carrots(Daucus carota)[J].Transgenic Research,18(6):911-999.
Sharma A K,Sharma M K.2009.Plant as bioreactors:Recent developments and emerging opportunities[J].Biotechnology Advances,27(6):811-832.
Starodubova,Preobrazhenskaia,Kuzmenko,2015.Rabies vaccines:Current status and prospects for development.[J].Molecular Biology,49(4):513-519.
Wang X H,Jiang D M,Yang D C.2015.Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay[J].Applied Biochemistry and Biotechnology,175(2):996-1006.
Wongarce A,Gonzálezortega O,Rosalesmendoza S.2017.Plant-made vaccines in the fight against cancer[J].Trends in Biotechnology,35(3):241-256.
Xu J F,Ge X M,Dolan M C.2011.Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures[J].Biotechnology Advances,29(3):278-299.
冯烨.2015.中国动物狂犬病流行病学研究[D].博士学位论文,中国人民解放军军事医学科学院,导师:涂长春pp.94.(Feng Y.2015.Epidemiology of animal rabies in China[D].Doctoral thesis.Academy of Military Medical Sciences,Chinese People's Liberation Army),Instructor:Tu C C,pp.94.)
李冰,顾劲乔.2017.中国狂犬病年会综述[J].中国工作犬业,(5):58-60.(Li B,Gu J Q.2017.Overview of 2017Rabies conference in china[J].Chinese Working Dog Industry,(5):58-60.)
孙喆,刘营,张明辉,等.2015.转OsDREB3基因大豆外源基因拷贝数的确定及标准分子的构建[J].甘肃农业大学学报,50(03):61-67.(Sun Z,Liu Y,Zhang M H,et al2015.Determination of the foreign gene copy number of OsDREB3 gene and the construction of standard molecules[J].Journal of Gansu Agricultural University,50(03):61-67.)
许倩茹,张二芹,郭军庆,等.2017.新城疫病毒F蛋白在水稻中的表达及检测[J].畜牧兽医学报,(6):1167-1172(Xu Q R,Zhang E Q,Guo J Q,et al.2017.Expression and detection of Newcastle disease virus F protein in rice[J].Journal of Animal Husbandry and Veterinary Medicine,(6):1167-1172.)
余笑波,崔海峰,俞晓平,等.2012.水稻淀粉分支酶基因(RBE4)作为转基因水稻基体标准物质的内标准基因的研究[J].农业生物技术学报,20(11):1234-1243.(Yu X B,Cui H F,Yu X P,et al.2012.Studies on rice starch branching enzyme(RBE4)as endogenous reference gene for the matrix reference material of transgenic rice(Oryza sativa L.)[J].Journal of Agricultural Biotechnology,20(11):1234-1243.)
张守峰,刘晔,赵敬慧,等.2012.从流行毒株的系统发生看中国目前狂犬病流行的主要特征[J].中国动物传染病学报,20(4):19-23.(Zhang S F,Liu Y,Zhao J H,et al.Epidemiological characterization of rabies virus isolates in china by phylogenetic analysis[J].Chinese Journal of Animal Infectious Diseases,20(4):19-23.)
Boothe J,Nykiforuk C,Shen Y,et al.2010.Seed-based expression systems for plant molecular farming[J].Plant Biotechnology Journal,8(5):588-606.
Delagneau J F,Perrin P,Atanasiu P.1981.Structure of the rabies virus:Spatial relationships of the proteins G,M1,M2 and N[J].Ann Inst Pasteur Virol,132(4):473-493.
Jiao W Q,Yin X P,Li Z Y,et al.2011.Molecular characterization of China rabies virus vaccine strain[J].Virology Journal,17(8):521-531.
Paul M,Ma K C.2011.Plant-made pharmaceuticals:Leading products and production platforms[J].Biotechnology&Applied Biochemistry,58(1):58-67.
Qiu J R,Xu Y,Yu F G.2011.Determining the copy number of exogenous gene in transgenic plant by SYBR green realtime quantitative PCR[J].Agricultural Science&Technology,12(6):829-831,835.
Rojas-Anaya E,Loza-Rubio E,Olivera-Flores M T,et al.2009.Expression of rabies virus G protein in carrots(Daucus carota)[J].Transgenic Research,18(6):911-999.
Sharma A K,Sharma M K.2009.Plant as bioreactors:Recent developments and emerging opportunities[J].Biotechnology Advances,27(6):811-832.
Starodubova,Preobrazhenskaia,Kuzmenko,2015.Rabies vaccines:Current status and prospects for development.[J].Molecular Biology,49(4):513-519.
Wang X H,Jiang D M,Yang D C.2015.Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay[J].Applied Biochemistry and Biotechnology,175(2):996-1006.
Wongarce A,Gonzálezortega O,Rosalesmendoza S.2017.Plant-made vaccines in the fight against cancer[J].Trends in Biotechnology,35(3):241-256.
Xu J F,Ge X M,Dolan M C.2011.Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures[J].Biotechnology Advances,29(3):278-299.