生长抑素基因疫苗pVAX1-TRX-SS的构建及对猪生长的影响  

Construction of somatostatin gene vaccine pVAX1-TRX-SS and its effect on pig growth

作　　者：黄志鹏 陈婷 孙加节[1] 习欠云[1] 史合群 张永亮[1] HUANG Zhi-peng;CHEN Ting;SUN Jia-jie;XI Qian-yun;SHI He-qun;ZHANG Yong-liang(National Pig Seed Engineering Center,Guangdong Provincial Key Laboratory of Animal Nutrition,College of Animal Science,South China Agricultural University,Guangzhou 510642,China;Guangzhou Chuang Wei Xi Wang Biological Technology Co.,Guangzhou 510000,China)

机构地区：[1]华南农业大学动物科学学院国家生猪种业工程中心广东省动物营养调控重点实验室,广东广州510642 [2]广州市创唯曦旺生物科技有限公司,广东广州510000

出　　处：《中国兽医学报》2018年第12期2360-2365,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2016YFD0500503);国家自然科学基金面上资助项目(31472163);广东省饲料产业创新体系基金资助项目(2017LM1121)

摘　　要：用带有生长抑素CDS(sequence coding for amino acids in protein)区的引物,以pET32a(+)中的TRX标记基因为模板,扩增获得生长抑素(somatostatin,SS)-硫氧还原蛋白(thioredoxin,TRX)基因,并将该段基因插入pVAX1质粒载体中,构建pVAX1-TRX-SS真核表达质粒,酶切和测序鉴定表明质粒构建正确。用Lipofectamine~2000Reagent将上述质粒转染CHO细胞进行瞬时表达,24h后提取细胞总蛋白,Western blot能检测到目的蛋白。选取60日龄去势公猪18头,随机分为3组,分别于半膜肌注射0mg(生理盐水),1mg和2mg pVAX1-TRX-SS质粒,生理盐水对照组为Ⅰ组,1mg和2mg组分别为Ⅱ和Ⅲ组,注射后用活体基因导入仪电击处理。结果显示,免疫后5周,Ⅱ组和Ⅲ组平均日增重分别比Ⅰ组提高13.92%(P<0.05)和11.40%(P>0.05),料重比分别降低了6.19%和3.33%(P>0.05)。免疫后2周,Ⅱ组血清中生长抑素含量显著低于Ⅰ组;Ⅲ组血清中生长抑素含量则在免疫后4周显著低于Ⅰ组。Ⅱ组生长抑素抗体阳性率于免疫后4周达到峰值80%,Ⅲ组则在免疫后4周达到80%并维持到免疫后6周。上述结果表明,pVAX1-TRX-SS真核表达质粒能在真核细胞中表达,免疫猪后使猪产生了生长抑素抗体,有效中和了内源性生长抑素,对猪的生长有一定促进作用。Somatostatin-thioredoxin fusion gene was amplified by the primer containing somatostatin CDS(sequence coding for amino acids in protein)region,and the template was TRX marker gene of pET32 a(+).Then,the fusion gene was inserted into the pVAX1 plasmid vector and pVAX1-TRX-SS eukaryotic expression plasmid was constructed.Enzyme digestion and sequencing showed that the plasmid were correctly constructed.Lipofectamine~2000 Reagent was used to transfect the plasmid into CHO cells for transient expression.After 24 h,the total protein of the cells was extracted,and Western blot could detect the target protein.Eighteen 60-day-old castrated boars pigs were randomly divided into three groups and respectively injected 0 mg(groupⅠ),1 mg(groupⅡ)and 2 mg(group Ⅲ)pVAX1-TRX-SS plasmid in semimembranous muscle.The pigs were electroporated with gene pulser transfection apparatus after intramuscular injection.Comparing with groupⅠ,the average daily gain in group Ⅱ and group Ⅲincreased by 13.92%(P<0.05)and 11.40%(P>0.05),and feed/gain both decreased by 6.19%and 3.33%(P>0.05)at the experimental period of forty-fifth days.2 weeks after immunization,the somatostatin in serum of groupⅡ was significantly lower than that groupⅠ.4 weeks after immunization,the somatostatin in serum of groupⅢ was significantly lower than group Ⅰ.4 weeks after immunization,the positive rate of somatostatin antibody in group Ⅱ reached 80%.In the group Ⅲ this rate reached 80%4 weeks after immunization and maintained to 6 weeks.These results showed that the pVAX1-TRX-SS plasmid can be expressed in eukaryotic cells.It stimulates the production of somatostatin antibody after immunization and effectively neutralized endogenous somatostatin,which promotes the growth of pigs.

关 键 词：生长抑素 电击处理 去势公猪 生长性能 

分 类 号：S816.73[农业科学—饲料科学] S852.52[农业科学—畜牧学]