Screening The LPL Gene For Mutations And Cloning And Expression Of Mouse ApoCⅡ

Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism, its major function is the hydrolysis of the core triglycerides of circulating chylomicrons and very low density lipoproteins. Apolipoprotein CⅡ is required for the enzymatic activity of LPL. The major deficiency of LPL and Apolipoprotein CⅡ can cause hypertriglyceridemia. Hypertriglyceridemia is the important risk factor of many diseases , such as, atherosclerosis and pancreatitis et al..This experiment is divided into two parts. Firstly, to screen the LPL gene for mutations in 203 Chinese population and to study the possible effects of the mutations on plasma lipids, the LPL gene (exon 4 and regulatory sequence) was examined by PCR-SSCP analysis. The PCR products showing abnormal pattern on SSCP were sequenced using dideoxy chain termination method. One mutation was found in exon 4 amplified fragment of a person with normolipidemia by PCR-SSCP and identified to be a single transition (C→T) at six bp upstream from acceptor splicing site of intron 3 by DNA-sequencing. none of the other mutations was found in this study. The transition (C→T) mutation in intron 3 of the LPL gene was generally considered to be one of the genetic risks for hyperglyceridemia. Up to day, all this mutation in references was only found in hypertriglyceridemia, so the discovery of the mutation in person with normolipidemia may be certainly valuable to study the possible effects of themutation.Secondly, the encoding gene of Apolipoprotein C II was amplified byRT-PCR using total RNA derived from mouse liver tissues as template. The amplified fragment was recombined into pUC18 plasmid. Recombinant vector pUC18-mouse ApoCⅡ(pUC18-mApoCⅡ) was obtained and then sequenced.The gene encoding for apolipoprotein CII obtained from pUC18-mApoC II was inserted into fusion expressing vector pGEX-3X. The sequencing of Apolipoprotein CII in recombined plasma pUC18-mApoClIand pGEX-mouseApoC II (pGEX-mApoC II) was the same to GenBank sequence. Furthermore, recombinant vector pGEX-mApoC II was transformed into E.coli DH5a. After the culturing conditions were optimized, fusion protein GST-mApoC II was obtained. Fusion proteins was purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion protein was characterized by SDS-PAGE and Western blotting, hi this experiment, the mouse Apolipoprotein CII was cloned and expressed successfully. The production of mouse ApoC II by genetic engineering may provide a nessasary basis to prepare antibodies of ApoC II and to study the possibility and effects of treatment of ApoC II to patients with hypertriglyceridemia.