猪脂蛋白脂酶基因片段的克隆及不同体重的表达差异

从杜长大母猪的肠系膜脂肪中提取基因组RNA，用RT-PCR扩增脂蛋白脂酶(lipoprotein lipase LPL)基因，获得1条约689 bp的片段，以pGEM-T Easy vector 为载体，将该基因片断克隆到大肠杆菌(Escherichia coli )DH5α中。从筛选的阳性克隆中分离出LPL基因，测定其序列。分析表明，该片段为LPL cDNA的部分序列，编码229个氨基酸组成的多肽。研究得到的基因片段与报道的猪脂肪组织中LPL cDNA部分序列同源性达到98%，氨基酸序列同源性达到99.1％。以LPL基因片段的克隆为基础，构建了优化的半定量RT-PCR法，以β-actin 为内标，研究不同体重猪脂肪组织LPL基因表达的差异。结果发现，从刚出生到30 kg，LPL基因表达呈上升趋势，在30～50 kg，LPL基因表达呈下降趋势，50～90 kg，LPL基因表达又呈上升趋势。

Cloning of Lipoprotein Lipase(LPL) Gene of Swine and the Difference of LPL Gene Expression at Different Avoirdupois Stages

Genome RNA was extracted from mesentery adipose of pig (Duroc Landrace Yorkshire) and lipoprotein lipase (LPL) mRNA was amplified using RT-PCR. A DNA fragment about 689 bp in length was obtained and the PCR product was cloned into pGEM-T vector. The LPL gene was isolated and sequenced from the screened positive clones. Result of sequence analysis showed that this fragment was the partial sequence of LPL cDNA and coded 279 amino acid residues. The gene homology of the obtained fragment compared with that of reported LPL gene sequence in adipose of porcine was up to 98%, and amino acid homology was 98.94%. Based on the LPL gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Using β-actin as inner control, the difference of LPL gene expression at different avoirdupois of swine was researched, which increased from new born to 30 kg, decreased from 30 to 50 kg and rose again at the stage of 50 to 90 kg.