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2025年4月5日 星期六
农业生物技术学报  2019, Vol. 27 Issue (2): 204-211    DOI: 10.3969/j.issn.1674-7968.2019.02.002
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
狂犬病病毒G蛋白在水稻中的表达及遗传稳定性鉴定
申雪静1, 张二芹1, 许倩茹3, 刘林科1, 万博1, 刘运超2, 孙亚宁2, 赵东2, 牛香香1, 邓瑞广2, 张改平1*
1 河南农业大学 牧医工程学院,郑州 450002;
2 河南省农业科学院 动物免疫学重点实验室,郑州 453002;
3 西北农林科技大学 动物医学院,杨凌 712100
Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability
SHEN Xue-Jing1, ZHANG Er-Qin1, XU Qian-Ru3, LIU Lin-Ke1, WAN Bo1, LIU Yun-Chao2, SUN Ya-Ning2, ZHAO Dong2, NIU Xiang-Xiang1, DENG Rui-Guang2, ZHANG Gai-Ping1*
1 College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
2 Key Laboratory of Animal Immunology/Henan Academy of Agricultural Sciences, Zhengzhou 453002, China;
3 College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China
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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。
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申雪静
张二芹
许倩茹
刘林科
万博
刘运超
孙亚宁
赵东
牛香香
邓瑞广
张改平
关键词 狂犬病病毒G基因转基因水稻荧光定量纯合子拷贝数    
Abstract: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 pMP3-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, and 313 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.
Key wordsRabies virus    G gene    Transgenic rice    Fluorescence quantification    Homozygote    Copy number
收稿日期: 2018-10-10     
ZTFLH:  S851.3  
基金资助:优质与功能型转基因水稻新品种培育(No.2016ZX08001006)
通讯作者: *,zhanggaiping20003@163.com。   
引用本文:   
申雪静, 张二芹, 许倩茹, 刘林科, 万博, 刘运超, 孙亚宁, 赵东, 牛香香, 邓瑞广, 张改平. 狂犬病病毒G蛋白在水稻中的表达及遗传稳定性鉴定[J]. 农业生物技术学报, 2019, 27(2): 204-211.
SHEN Xue-Jing, ZHANG Er-Qin, XU Qian-Ru, LIU Lin-Ke, WAN Bo, LIU Yun-Chao, SUN Ya-Ning, ZHAO Dong, NIU Xiang-Xiang, DENG Rui-Guang, ZHANG Gai-Ping. Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability. 农业生物技术学报, 2019, 27(2): 204-211.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.02.002     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I2/204
 
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