Protection of Mice Against Challenge with Foot-and-Mouth Disease Virus by Immunization with vp1 Gene Expressed Transgenic N·Benthamiana

WANG Bao-qin; ZHANG Yong-guang; WANG Xiao-long; WANG Yong-lu; PAN Li; WANG Wen-xiu; DONG Jin-jie; LV Jian-liang

PDF
Cite
Share

facebook

twitter

google

linkedin

April 2005

WANG Bao-qin, ZHANG Yong-guang, WANG Xiao-long, WANG Yong-lu, PAN Li, WANG Wen-xiu, DONG Jin-jie and LV Jian-liang. Protection of Mice Against Challenge with Foot-and-Mouth Disease Virus by Immunization with vp1 Gene Expressed Transgenic N·Benthamiana[J]. Virologica Sinica, 2005, 20(2): 140-144.

Citation: WANG Bao-qin, ZHANG Yong-guang, WANG Xiao-long, WANG Yong-lu, PAN Li, WANG Wen-xiu, DONG Jin-jie, LV Jian-liang. Protection of Mice Against Challenge with Foot-and-Mouth Disease Virus by Immunization with vp1 Gene Expressed Transgenic N·Benthamiana .VIROLOGICA SINICA, 2005, 20(2) : 140-144.

FMDV vp1基因在烟草中表达及转基因烟草的免疫效果

王宝琴 ^1,2 ,
张永光 ^2* ,
王小龙 ¹ ,
王永录 ² ,
潘丽 ² ,
王文秀 ² ,
董金杰 ² ,
吕建亮 ²

1.
1.南京农业大学动物医学院 2.甘肃兰州中国农业科学院兰州兽医研究所

摘要

通过三亲杂交法,构建克隆有阿克苏(Akesu/58) O 型口蹄疫病毒vp1 基因的双元表达载体pBin FMDVVP1。采用农杆菌介导法转化NC89烟草叶盘,经卡那霉素筛选,共获得49株抗性植株。对抗性植株总DNA进行目的基因的PCR检测,有40株阳性植株。对阳性植株总RNA进行目的基因的RT PCR检测,有21株阳性植株。将7株ELISA和Western blot检测阳性植株叶片提取物分别与弗氏佐剂乳化,在0、15、30 和45d 腹膜腔接种Balb/C小白鼠,于第4次免疫后第9d进行血清抗体检测;第12d用104SM50 LD的同源强毒进行攻击;攻毒后24h采血,通过乳鼠病毒血症试验判定攻击Balb/C小白鼠的发病和保护情况。结果表明:双元表达载体pBin FMDVVP1构建正确;vp1基因转入NC89烟草并获得表达;7组中有2组Balb/C小白鼠血清抗体呈阳性,攻毒保护率分别为100%和63%。证明2株转基因烟草表达的VP1蛋白具有较好的免疫原性,所免疫的2 组Balb/C小白鼠对同源强毒攻击有一定的抵抗能力。
- 口蹄疫病毒
- , 表达
- , 烟草
- , 免疫
- , 抗体
- , Balb/C小白鼠

Protection of Mice Against Challenge with Foot-and-Mouth Disease Virus by Immunization with vp1 Gene Expressed Transgenic N·Benthamiana

1.
1. Col lege of Veterinary Medicine , N anj ing A gricultural Universit y , N anj ing 210095 , China

Abstract

A binary plasmid was constructed by triparental mating, which contained the vp1 gene of foot-and-mouth disease virus strain Akesu/58 serotype O. The Nicotiana Benthamiana (NC) leaves were transformed by Agrobacterium tumefaciens-mediated method with the binary expression plasmid. After selected by kanamycin, forty-nine resistant lines were obtained. Forty lines were positive through PCR with total DNA and twenty-one lines were positive through RT-PCR with total RNA from fresh leaves tissue. The leave extracts of seven positive lines analyzed with ELISA and western-blot were respectively emulsified in Freund′s adjuvant and the Balb/C mice were immunized intraperitoneally at days 0,15,30 and 45. After the last inoculation, the sera antibody were analyzed at 9 days and the mice were experimentally challenged intraperitoneally with 10~4 suckling mouse 50% lethal doses (SM_(50)LD) of FMDV at 12 days. Protection was determined by the absence of viremia at 24h post-inoculation. ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">Viremia was tested by intradermal in ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">intradermal inoculation of perip heral blood into suckling mice. These results showed that the const ructed binary expression plasmid was functionary ; vp1 gene was transformed into NC plants and expresed affirmatively ; the structure protein VP1 expressed in two transgenic plants lines may possibly be used as a source of antigen , the inoculated Balb/C mice developed a specific antibody response ; the protection against FMDV challenge in two groups was 100 % and 63 % respectively.
- Foot-and-mouth disease virus
- , Expression
- , Nicotiana benthamiana
- , Immune Antibody
- , Balb/C mice

References
Proportional views

PDF

Article Metrics

Article views(4236) PDF downloads(916) Cited by(0)

Proportional views

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Protection of Mice Against Challenge with Foot-and-Mouth Disease Virus by Immunization with vp1 Gene Expressed Transgenic N·Benthamiana

1. 1. Col lege of Veterinary Medicine , N anj ing A gricultural Universit y , N anj ing 210095 , China

Abstract: A binary plasmid was constructed by triparental mating, which contained the vp1 gene of foot-and-mouth disease virus strain Akesu/58 serotype O. The Nicotiana Benthamiana (NC) leaves were transformed by Agrobacterium tumefaciens-mediated method with the binary expression plasmid. After selected by kanamycin, forty-nine resistant lines were obtained. Forty lines were positive through PCR with total DNA and twenty-one lines were positive through RT-PCR with total RNA from fresh leaves tissue. The leave extracts of seven positive lines analyzed with ELISA and western-blot were respectively emulsified in Freund′s adjuvant and the Balb/C mice were immunized intraperitoneally at days 0,15,30 and 45. After the last inoculation, the sera antibody were analyzed at 9 days and the mice were experimentally challenged intraperitoneally with 10~4 suckling mouse 50% lethal doses (SM_(50)LD) of FMDV at 12 days. Protection was determined by the absence of viremia at 24h post-inoculation. ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">Viremia was tested by intradermal in ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">intradermal inoculation of perip heral blood into suckling mice. These results showed that the const ructed binary expression plasmid was functionary ; vp1 gene was transformed into NC plants and expresed affirmatively ; the structure protein VP1 expressed in two transgenic plants lines may possibly be used as a source of antigen , the inoculated Balb/C mice developed a specific antibody response ; the protection against FMDV challenge in two groups was 100 % and 63 % respectively.