因传染性胃肠炎死亡的转基因克隆仔猪的临床病理分析和初步分子鉴定  

Clinic,Pathological Anatomy Symptoms and Preliminary Molecular Identification of Cloned Transgenic Pigs Dying of Transmissible Gastroenteritis

作　　者：赵雪艳[1] 洪渊[1] 赵科伟 王小鹏[1] 杨强[1] 王成斌[1] 何小芳[1] 邱青松[1] 程玉柱[1] 崔磊磊[1] 任军[1] 幸宇云[1] 

机构地区：[1]江西农业大学动物生物技术国家重点实验室培育基地,江西南昌330045

出　　处：《江西农业大学学报》2015年第3期504-511,共8页Acta Agriculturae Universitatis Jiangxiensis

基　　金：转基因生物新品种培育重大专项(2011ZX08006-005;2013ZX08006-005);江西省科技支撑计划(20111BBF60018)项目

摘　　要：观察记录6头因传染性胃肠炎死亡的转基因克隆大白仔公猪的临床和病理症状,比较这些猪和普通大白猪多个部位器官的脏器系数;通过常规PCR和Long PCR对转基因仔猪所导入的外源载体p EF-BMPRIB(CDS)-Neo进行扩增,利用实时荧光定量PCR(RT-PCR)绝对定量的方法检测外源载体中标记基因NEO的拷贝数。结果显示:患病死亡的转基因个体均表现出腹泻及粪便异常的症状,部分个体伴有发烧、咳嗽等不同症状,解剖后发现所有个体均表现出小肠肿胀和胃内充有凝乳,部分个体伴有小肠充血的症状;脏器系数分析结果表明,转基因克隆公猪与普通大白公猪之间除肾脏系数存在差异(P<0.05)外,其余脏器系数差异均不显著(P>0.05);分子鉴定结果显示,6头克隆猪均为转基因阳性个体,但外源载体序列在不同的部位出现丢失或断裂的现象,其中4头在外源载体的关键启动子和CDS区出现断裂或丢失,外源载体中NEO的拷贝数在6头转基因猪克隆猪中分别为1、2、2、3、4、5。综上所述,因传染性胃肠炎死亡的转基因克隆仔猪在临床和病理症状方面除表现出该病的典型特症外无其它异常,且这些仔猪和普通大白仔猪之间的脏器系数仅有微小差异,研究中没有证据表明转基因克隆技术导致克隆仔猪出现明显的异常;分子鉴定表明该批转基因克隆猪均为阳性个体,但外源基因并未完整转入宿主基因组中。In this study, the clinical and pathological symptoms of 6 cloned transgenie Large White dying of transmissible gastroenteritis were recorded, and the organ coefficients of multiple tissues between these trans- genic pigs and normal Large White pigs were compared. Conventional PCR and Long PCR were performed to amplify the exogenous pEF-BMPRIB （CDS）-Neo vector, and quantitative real-time PCR （qRT-PCR） was used to detect the copy numbers of exogenous NEO by absolute quantification assay in these transgenic pigs. The results showed that all of the sick cloned transgenic piglets exhibited diarrhea and stool abnormality, and some had fever and cough.The dissection revealed that all these pigs had intestine swelling and milk curd in stomach, and some piglets showed congestion in small intestine. Organ coefficient analyses indicated that no sig- nificant difference was found in the detected tissues （P〉0.05） except the kidney （P〈0.05） between transgen- ic and non-transgenic pigs.Molecular identification revealed that 6 cloned transgenic pigs were transgenic posi- tive individuals,whereas with loss or disruption of exogenous vector at different positions. Of the 6 transgenic pigs,4 lost or had disruptions in the promoter or CDS regions.The copy numbers of exogenous NEO in the 6 transgenic pigs were 1,2,2,3,4 and 5. In conclusion, cloned transgenic Large White piglets dying of transmissi- ble gastroenteritis showed no other abnormalities except the typical clinic and pathological anatomy symptoms of this disease.Only slight difference was found in the organ coefficients between cloned transgenic and normal Large White piglets.There was no evidence that transgenic or cloning techniques led to significant changes in recipient piglets.Molecular identification indicates that these cloned pigs are transgenic positive individuals, but the exogenous gene did not completely integrate into the host genome.

关 键 词：转基因克隆 大白猪 分子鉴定 临床和病理症状 脏器系数 

分 类 号：S814.8[农业科学—畜牧学]