pGH和IGF-Ⅰ双基因共表达载体的构建及转双基因猪的获得与检测

旨在构建可高效表达pGH基因和IGF-Ⅰ基因的双基因共表达载体,制备转双基因(pGH+IGF-Ⅰ)猪,以期探索pGH基因和IGF-Ⅰ基因对猪生长发育的影响,为节粮型高瘦肉率新品种猪的培育奠定理论基础。从长白猪耳样中提取总RNA,经反转录RT-PCR获得pGH基因不含终止密码子的编码序列和IGF-Ⅰ基因完整的编码序列,经酶切连接克隆至pc DNA3.1(+)真核表达载体上,构建pc DNA3.1(+)-pGH-IGF-Ⅰ双基因共表达载体。将其转染PK15细胞,Q-PCR检测2个目的基因在PK15细胞中的表达情况。将构建的双基因共表达载体用纳米材料包裹后转染长白猪精子,采用精子载体法制备转双基因猪。PCR及测序鉴定转双基因阳性个体,Q-PCR检测2个目的基因在转双基因猪体内的表达情况。PCR及测序鉴定追踪检测转双基因猪体内pGH基因和IGF-Ⅰ基因的稳定情况。RT-PCR及测序结果表明,成功克隆了长白猪的pGH基因和IGF-Ⅰ基因的编码序列。酶切和测序分析表明成功构建了双基因真核共表达载体,转染PK15细胞后,Q-PCR检测表明,pGH基因和IGF-Ⅰ基因均在mRNA水平成功表达。母猪妊娠获得13头仔猪,经PCR及测序检测,其中4头仔猪为转双基因阳性,转双基因阳性率为30.76%。Q-PCR检测外源pGH基因与IGF-Ⅰ基因在转双基因猪体内成功表达。1~7月龄均可检测到外源pGH基因与IGF-Ⅰ基因,证明2个外源基因在转双基因猪体内稳定存在,并未随着生长而丢失。在转双基因公猪的精液中均能检测到2个外源基因,证明外源基因存在稳定传代的可能。

Construction of Eukaryotic Co-expression Plasmid Carrying pGH and IGF-Ⅰ Gene and It′s Transformation in Landrace

In order to explore the effects of pGH and IGF-Ⅰ on the growth and development of Landrace,eu-karyotic co-expression plasmid containing pGH and IGF-Ⅰ was constructed and transfected into Landrace,which will lay the foundation for the cultivation of new breed of feed-saving grain pigs.Total RNA was extracted from the ear tissue of Landrace pig,and the coding sequence of pGH without termination codon and complete coding sequence of IGF-Ⅰ were amplified by RT-PCR and cloned into the pcDNA3.1 (+)eukaryotic expression vector after verified by sequencing.The recombinant plasmid was verified by sequencing and enzyme digestion and then transfected into PK15 cells.Expression of the two genes in PK15 cells was detected by Q-PCR.Transgenic pigs were prepared with sperm-mediated transformation after the sperm were encapsulated by nanometer material.Transgenic pigs were iden-tified by PCR and sequencing.The expression of the two target genes were detected by Q-PCR.Stability of transgene was detected by PCR and sequencing aged from 1 month to 7 month.RT-PCR and sequencing results showed that the coding sequences of pGH and IGF-Ⅰ of Landrace were successfully cloned.Digestion and sequencing analysis showed the eukaryotic co-expression vector containing pGH and IGF-Ⅰ was successfully constructed,and Q-PCR a-nalysis showed that pGH and IGF-Ⅰ were successfully expressed at the mRNA level after transfected into PK15 cells.As a result,13 young piglets were born,and 4 of them were positive for both two genes detected by PCR and sequencing.As a result,the positive rate was 30.76%.Q-PCR results showed that exogenous pGH and IGF-Ⅰ were successful expressed in transgenic pigs.Exogenous pGH and IGF-Ⅰ could be detected in transgenic positive individ-uals from aged 1 month to 7 month,which proved the two exogenous genes were stable in transgenic pigs and were not lost in growth process.Exogenous pGH and IGF-Ⅰ could be detected in sperm of transgenic boars showed that exogenous pGH and IGF-Ⅰ may be passaged stable.