MLC₂-chymase Fusion Gene Clone, Expression In Cultured Cell And Transgenic Mice Production

Chymase is a kind of serine proteinase. It can highly specifically convert angiotensin I to angiotensin II.It was reported that in human left ventricule,over 75% angiotensin II converting enzyme activity can not be inhibited by angiotensin I converting enzyme inhibitor, but can be inhibited by serine proteinase inhibitor(mainly chymase activity in vitro). So chymase may play an important role in human heart. In mouse heart, only minor component(15%) of the angiotensin II forming enzyme activity come from chymase, so we can choose mice as laboratory animals and create a transgenic mice model harboring human heart chymase gene to, in vivo, investigate the expression manner of the human heart chymase gene, understand its structure and function , moreover elucidate the relationship between the expression and regulation of chymase gene and hypertension, cardiac hypertrophy and other cardiovascular diseases.MLC2 (myosin light chain 2) is a regulatable protein by phosphorylating in heart. α 1^- adenergic agonists can stimulate overexpression of the gene, so they can regulate the expression of chymase gene fused with MLC2 promoter efficiently. 1. The clone of MLC₂-chymase fusion gene.pCHI(containing 6kb human heart chymase gene) plasmid DNA was linearized by restriction endonuclease BstE II and deleted by nuclease BAL31. A series of structural chymase gene clones, which kept different length of promoter sequence, were obtained. The four suitable chymase structure gene clones (P81,P85,P87 and P88) were selected from the series of clones by sequencing. The fragments of 3.2kb structural chymase gene were recovered by low melt agarose after digesting the clones of suitable structural chymase gene with SpeI and KpnI and subsequently inserted into plasmid MLCSVOA(containing 250bp sequence of rat heart MLC2 promoter) which was digested by SpeI and KpnI, so that we can get the MLC₂-chymase fusion gene clones .The suitable clones (M81,M85,M87 and M88) of MLC₂- chymase fusion gene were selected through analysis...