The Expression Of Human Ubiquitious Mitochondrial Creatine Kinase In Escherichia Coli And Preparation Of The Specific Antibody Of UMtCK

Creatine kinase (creatine N-phosphotransferase;CK;EC 2.7.3.2) is the key enzyme of energy metabolism and catalyzes the reversible exchange of high-energy hposphoryl between ATP and creatine phosphate (PCr) . There are five isoenzyme in vivo , cytoplasmic CKs (BB-,MB-,and MM-CK) and mitochondria CK ( uMtCK and sMtCK ). Cytoplasmic CKs , mitochondria CKs ,creatine and PCr conform the system of energy buffering and transportation ,namely CK/PCR circuit which play an important role in energy metabolism of cells and tissues . Mitochondria CK , found in 1964 , has different structure , location , activity from cytoplasmic CKs although they both catalyze a same reaction of biochemistry . It causes a great interest in many researchers recent years because overexpression of uMtCK is found in malignant tumors with especially poor prognosis and it maybe plays an important role in interaction with some pore protein of mitochondrial membrane . The objective of this research is to get uMtCK fusion protein for next studing and analysing the cause of overexpression of uMtCK in tumor patients and evaluating the possibility that use uMtCK as serum marker of tumor . we have done the works as follows for the objective : 1) The coding sequence of uMtCK gene was cloned and inserted into plasmid to construct a prokaryotic expression vertor pQE30- uMtCK . 2) uMtCK fusion protein produced in E.coli M15 was induced ,expressed , purified and evaluated. 3) An antibody agansist uMtCK was perpetrated by immuned rabbit and the expression of uMtCK in tissue of 51 patients with gastric carcinoma or cancer of colon was detected by immunohistochemistry . Theresult of our study indicated that : l)The cDNA sequence of uMtCK were obtained successfully by RT-PCR from the cDNA of . Hela cell and then inserted into pQE30 to construct a recombinant expressive vector pQE30-uMtCK . 2) Upon the induction of IPTG, uMtCK fusion protein was expressed in E.coli M15. The fusion protein was purified with Ni-chelated affinity chromatography . The purified recombinant uMtCK produced in E. coli showed a partly enzyme activity. 3) With the purified recombinant uMtCK protein as immunogen, antiserum specific to uMtCK were havested from New Zealand rabbits and the Ouchterlony test and ELISA showed that the liters are 1:16 and 1:8000 respectively.The antiserum be used to detect the expression of uMtCK in tissue of 51 patients with gastric carcinoma or cancer of colon by immunohistochemistry . The result show that the positive rate (76.5%)of the expression of uMtCK in tissue of gastric carcinoma or cancer of colon detected with preparated antibody is higher significantly than that detected with purchased antibody of uMtCK and sMtCK (52.9% and 45.1% , respectively).