ABSTRACT
BACKGROUND: Studies have shown that alanine (A) to threonine (T) substitution at codon 54 of intestinal fatty acid-binding protein (FABP2) in different populations is associated with dyslipidemia and other characteristics of metabolic syndrome.OBJECTIVE: To investigate the frequency of encoding 54Ala/Thr (A/T) single nucleotide polymorphism in the FABP2 in middle-aged and old people, and explore the association between 54T FABP2 and plasma lipids.DESIGN: A case-controlled analysis. SETTING: Department of Biochemistry, Hebei North University and Department of Clinical Laboratory, the 251 Hospital of Chinese PLA.PARTICIPANTS: 469 physical examinees were selected from the Medical Examination Center, the 251 Hospital of Chinese PLA between October 2003 and April 2005. The subjects included 217 males with mean age of (56±10) years, and 252 females with mean age of (55±13) years. Only people with normal liver and kidney function, and with no blood relation were recruited. The informed consent to this study was obtained from all subjects. The experiment was admitted by Hospital Ethics Committee. METHODS: ①After fasting for 12 hours, automatic analyzer (Olympus AU 6400) was adopted to measure plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apoprotein A1(Apo A1) and Apo B levels. ②1 mL venous blood was extracted and immediately mixed with anti-coagulants containing citric acid, natrium citricum and glucose. White blood cells were separated and genomic DNA was isolated using standard methods with proteinase K digestion and phenol/chloroform purification. The genotype distribution frequency in each group was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). MAIN OUTCOME MEASURES: ①Plasma TC, TG, HDL-C, LDL-C, Apo A1and Apo B levels; ②Distributions of FABP2 genotypes at codon 54. RESULTS: ①The genotype frequencies of A/A, A/T, T/T were 0.48, 0.42, and 0.10 in males, and 0.44, 0.46, and 0.10 in females, respectively. The allelic frequency of point mutant 54Thr in FABP2 gene was 0.31 in males and 0.33 in females, respectively. There was no difference between males and females (χ2=0.47, P ＞ 0.05). ②The LDL-C and Apo B concentrations in fasting plasma of males with 54T allele were significantly higher than those with 54A allele (P ＜ 0.05). The TC and LDL-C concentrations in fasting plasma of females with 54T allele were significantly higher than those with 54A allele (P ＜ 0.05). CONCLUSION: In the middle-aged and old populations, the frequency of encoding 54Ala/Thr polymorphism in FABP2 gene is not correlated with gender, but with high lipoprotein profile.
ABSTRACT
Objective To clone the reconstructed Hc gene of botulinum neurotoxin type A(BoNT/AHc)and to explore the soluble expression of the reconstructed gene in E.coli.Methods The gene of Hc fragment was synthesized by replacing rare codons with frequent ones in E.coli,while the components of amino acids didn't change,and the contents of AT decreased from 76.4 % to 57.3%.The reconstructed gene was then cloned into the prokaryotic expression vector pQE-60.The recombinant plasmid pQE-60Hc was introduced into E.coli Origami(DE3)that was induced to express the protein.The soluble expression products were then detected by Western Blot.Finally the expressed product of recombination plasmid pQE-60Hc was analyzed with SDS-PAGE after purification through Ni-NTA column.Results The reconstructed Hc gene of BoNT/AHc was amplified by PCR.The expression vector pQE-60Hc was constructed successfully with BamH Ⅰ and Nco Ⅰto ingest both BoNT/AHc and vector pQE-60.Reconstructed gene could be expressed effectively in E.coli in soluble form.The molecular weight of expressed product of recombination plasmid pQE-60Hc analyzed by SDS-PAGE was 52 000,which was the same as anticipated.And the soluble expression product accounted for to 11.5 % of the total bacterial protein.Western blot assay showed that the expression product could bind to specific antibody agent BoNT/A.Conclusion The expression vector has been constructed and the reconstructed gene was expressed successfully and effectively in E.coli,which may provide a foundation for further study on antitoxin and vaccine.