| Human urinary trypsin inhibitor (hUTI) is a Kunitz-type protease inhibitor, which contains two Kunitz-type trypsin inhibitor domains. It exhibits an inhibitory activity against proteases including trypsin, chymotrypsin, plasmin and neutrophil elastase. The molecule is synthesized in hepatocytes, where it is linked to one or two heavy chains in the Golgi apparatus and forms inter-α-trypsin inhibitor (IαI) and pre-α-trypsin inhibitor (PαI). There is a significant increase in the urine UTI concentration in patients with inflammation or cancers. UTI may reduce the production of inflammatory factors such as TNF, IL-1 and IL-6. It can increase the stability of lysosome membrane and inhibit tumor cell invasion. It has also protective effect on ischemia/reperfusion injury of organs and has been widely used as a drug for patients with acute inflammatory disorders such as disseminated intravascular coagulation, shock and pancreatitis. It could be useful therapeutic tools to cancers and ischemia/reperfusion injury. At present, the protein of UTI is isolated from human urine with traditional chemical method. The source of urine is limited and maybe contain viruses, which must be treated with complicated virus-inactivation procedure. at the most, the protein is available only in low amounts. So we are to construct an expression system of recombinant hUTI and seek to produce recombinant protein with genetic engineering technology. UTI is encoded by α1-microglobulin/bikunin precursor (ambp) gene, which also encodes for α1-microglobulin (α1M), an immunosuppressive lipocalin. Despite they lack of any structural or functional relationship, α1M and UTI originate from AMBP cleavage by a furin-like protease that releases the two mature molecules. The expression of ambp gene is thought as strictly liver-specific in human, though ambp mRNA was detected also in pancreas, stomach, kidney and muscle in pig, mouse and rat. In this study, the ambp mRNA levels in human embryo liver, kidney and pancreas were examined by RT-PCR. The ambp mRNA was detected only in liver but not in other two tissues, which confirmed the liver-specification of the ambp mRNA expression. Then the ambp cDNA sequence available from the liver total RNA was used to construct the recombinant hUTI expression system as follows. 1. Cloning of ambp cDNA sequence We first obtained the cDNA sequence encoding AMBP by RT-PCR, in which the template RNA derives from the human embryo liver. Then the purified PCR production was cloned to pMD18-T vector. The results of sequencing showed that the recombinant cloning vector pMD18-T-ambp was constructed correctly. 2. Construction of recombinant hUTI expression system (1) Construction of hUTI expression vector pPICZα-huti With the recombinant plasmid of pMD18-T-ambp as template, the sequence encoding hUTI was obtained by PCR. The endonuclease sites of XhoI and XbaI were added into the two ends of interested sequence. Cloning the sequence to pPICZ αvector after being digested with those two endonucleases. The results of sequencing demonstrated that the construct was correct.
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