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| 密码子优化的PRRSV GP5基因DNA疫苗在鼠体内免疫效力的评价 | |
| 引用本文： | 侯艳红,周艳君,闫丽萍,田志军,杨汉春,童光志.密码子优化的PRRSV GP5基因DNA疫苗在鼠体内免疫效力的评价[J].中国预防兽医学报,2007,29(8):605-610. |
| 作者姓名： | 侯艳红 周艳君 闫丽萍 田志军 杨汉春 童光志 |
| 作者单位： | 1. 中国农业大学,农业部预防兽医学重点实验室,北京,100094;中国农业科学院,哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江,哈尔滨,150001 2. 中国农业科学院,哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江,哈尔滨,150001 3. 中国农业大学,农业部预防兽医学重点实验室,北京,100094 |
| 基金项目： | 国家重点基础研究发展计划(973计划);国家自然科学基金 |
| 摘 要： | 为了提高表达含有猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）GP5基因DNA疫苗的死疫效应,本研究通过人工合成的方法将GP5基因的密码子改造为猪体嗜好的密码子,并将其作为免疫原基因,连同野生型GP5基因,分别插入表达载体pIRES1neo中,构建重组质粒pIR-optiGP5和pIR-GP5.将这两种质粒分别转染293T细胞,转染后48 h,采用间接免疫荧光和Western blot方法检测GP5基因的体外表达情况,结果两种检测方法显示重组质粒pIR-optiGP5的蛋白表达量明显多于pIR-GP5。为了评价DNA免疫质粒pIR-optiGP5的免疫效果,选取30只6～8周龄雌性BALB/c小鼠,将pIRES1neo、pIR-GP5、pIR-optiGP5分别以100μg/只剂量,进行肌肉多点注射,共免疫3次,每次间隔2周,同时,利用间接免疫荧光技术、流式细胞技术、淋巴细胞特异性增殖试验等方法检测其体液免疫和细胞免疫水平。结果显示：DNA免疫质粒pIR-optiGP5在二免后即可检测到荧光抗体,而pIR-GP5在三免后才能检测到;且发现用pIR-optiGP5质粒免疫小鼠,其CD4^＋、CD8^＋ T淋巴细胞百分数及特异性刺激指数均高于pIR-GP5免疫小鼠,推测经过密码子优化的GP5基因其蛋白在小鼠体内得到了较高水平的表达,并诱导了较强的免疫应答,这为进一步研究和设计有效的PRRSV DNA疫苗提供了新的思路。 |
| 关 键 词： | 猪繁殖与呼吸综合征病毒 GP5基因 密码子优化 DNA免疫 |
| 文章编号： | 1008-0589（2007）08-0605-06 |
| 修稿时间： | 2007-01-31 |
Immunization of mice with DNA vaccines encoding PRRSV GP5 gene with optimized codon usage |
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| HOU Yan-hong,ZHOU Yan-jun,YAN Li-ping,TIAN Zhi-jun,YANG Han-chun,TONG Guang-zhi.Immunization of mice with DNA vaccines encoding PRRSV GP5 gene with optimized codon usage[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(8):605-610. | |
| Authors: | HOU Yan-hong ZHOU Yan-jun YAN Li-ping TIAN Zhi-jun YANG Han-chun TONG Guang-zhi |
| Institution: | 1. Key Laboratory of Preventive Veterinary Medicine of Ministry of Agriculture, China Agricultural University, Beijing 100094, China; 2. National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China |
| Abstract: | To improve the immunogenicity of DNA vaccines encoding the GP5 gcnc of PRRSV,we generated a synthetic PRRSV GP5 sequence in which most wild-type codons were replaced with codons from highly expressed pig genes(optiGP5).The optiGP5 gone and wild-type gene(GP5)were inserted into pIRESlneo to generate pIR-optiGP5 and pIR-GP5 vectors,respectively. Expressions of pIR-optiGP5 and pIR-GP5 gene in 293T cells were compared by Western blot and immunofluorescence assay.The immunogenicity of the two constructs was evaluated in BALB/e mice following intramuscular inoculation,by measuring the im- mune responses including GP5 specific antibody production,lymphocyte proliferation and production of CD4~ ,CD8~ cell in periph- eral blood lymphocytes.The results showed that expression of GP5 in vitro was considerably increased in comparison to that of the respective wild-type sequence in the same vector.In BALB/c mice,DNA immunization with pIR-optiGP5 resulted in an earlier GP5 antibody production,a significantly enhanced stimulation index and a marked increase in percentage of CD4~ ,CD8~ T cells in comparison to pIR-GP5 immunization.These results suggested a direct correlation between GP5 expression levels and the im- mune responses.Thus,synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy of DNA vac- cination. |
| Keywords: | porcine reproductive and respiratory syndrome virus GP5 gene codon optimization DNA immunization |
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