Screening Of The Lipoprotein Lipase Gene For Mutations And The Cloning And Expression Of The Human Apolipoprotein CⅡ Gene

Lipoprotein lipase (LPL) is a critical enzyme of catabolic metabolism in lipoproteins. Its major function is the hydrolysis of the core triglycerides of circulating chylomicrons and very low density lipoproteins. The mutations in the LPL gene may lead to the change of LPL activities and masses. Apolipoprotein Câ…¡ (ApoC â…¡) serves as a physiological activator of lipoprotein lipase. When ApoC â…¡ is absent or nonfunctional, the function of LPL is severely impaired. The decrease in LPL activity in the postheparin plasma of patients with ApoC â…¡ deficiency is corrected by the addition of normal ApoC â…¡ in vitro, or by the intravenous infusion of ApoC â…¡.The research is divited into two parts. The first part is the screening of the lipoprotein lipase gene for mutations. Mutations of the LPL gene (exon 2, exon 3 and intron-exon boundaries) were examined with polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP), and the amplified products showing abnormal pattern on SSCP were sequenced using dideoxy chain termination method. One mutation was found in 203 Chinese people. It was a heterozygote for CTG→TTG change in codon 103 of the LPL gene verified by the DNA sequencing. The mutation was one samesense mutation (Leu¹⁰³â†’Leu) in exon3, and wasn't reported previously. The mutation has been submitted to GenBank and one accession number (DQ083390) was obtained.The second part is the cloning and expression of the ApoC â…¡ encoding sequence. In this experiment, human genomic DNA was extracted from white blood cells, and it served as the template for PCR to amplify ApoC â…¡ gene. The mature peptide of Apolipoprotein Câ…¡ is encoded by only two exons, exon 3 and exon 4. The two exons were amplified respectively, then they were spliced together in vitro to obtain the DNA sequence encoding the mature peptide ofApoC II, which is a 246bp DNA fragment. The amplified fragment was inserted into cloning vector pUC18. When the sequence was confirmed to be correct by the DNA sequencing, the gene was recombined into fusion expressing vector pGEX-3X, and then the recombinat expressing plasmid pGEX-3X/humanApoC II (pGEX-3X/hApoC II) was constructed. Furthermore, recombinant vector pGEX-3X/hApoC II was transformed into E. coli DH5a. After the culturing conditions were optimized, the best condition was selected to express the fusion protein. Fusion protein was easily purified from bacterial lysates by affinity chromatography through Glutathione Sepharose 4B. The purified fusion protein was identified by the SDS-PAGE and Western blotting, and it was confirmed to be immunogenicity. The experiment provides a necessary basis for the preparation of bioactive mature peptide of ApoC II by genetic engineering. The production of the fusion protein may make it easy to prepare the antibodies of ApoC II and the bioactive mature peptide of ApoC II make it possible to study the possibilities and effects of ApoC II in treating the hypertriglycerolimia.