Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Long...Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.展开更多
With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve ma...With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve many racemic alcohols and esters. A number of results have also been reported about the resolution of amino acids in organic solvents. For example, DL-phenylalanine methyl ester was resolved by α-chymotrypsin in toluene/water biphasic system; in fact, this reaction was carried, out in water;transesterification of DL-phenylalanine propyl ester and methanol was catalyzed by α-chymotrypsin in methanol, and the enzyme used was dissolved in water and the展开更多
基金support provided by the 973 Program of China (2004CB117500)
文摘Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.
文摘With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve many racemic alcohols and esters. A number of results have also been reported about the resolution of amino acids in organic solvents. For example, DL-phenylalanine methyl ester was resolved by α-chymotrypsin in toluene/water biphasic system; in fact, this reaction was carried, out in water;transesterification of DL-phenylalanine propyl ester and methanol was catalyzed by α-chymotrypsin in methanol, and the enzyme used was dissolved in water and the
