Gene Cloning Of Nattokinase And Its Pharmacokinetics Studying In Rabbits

Cloning and sequencing analysis of gene encoding Nattokinase of Bacillus Subtilis (natto) were tested in this paper. Bacillus Subtilis (natto) genetic DNA was prepared with CTAB method. The purpose gene was gotten by PCR, then was ligated into pUCm-T vector with the help of T4 DNA lagase and transformed it into E.coli DH5αcompetent cell. Selecting the white colonies on the agar containing AMP and IPTG+X-Gal, and extracting its plasmid rapidly, identifying the recombinant plasmid. Then the plasmid of positive bacterium was extracted, PCR amplification and sequence analysis demonstrated that the 1278 bp Nattokinase gene including all of ORF (Opening Reading Fragment) was gotten. Which is compared with Nattokinase gene reported in genebank, there is only a base A changed into G allordingly, the code changed into GAG from GAA, but the amino acid that it encoded both is Glu. Other sequence, except those not being tested, is same as Nattokinase gene reported in literature cloning of Nattokinase gene provide the foundation that this enzyme becoming a gene engineering medicine.The pharmacokinetics of Nattokinse in rabbits was studied by ELISA.The objective is to set up a method to measure the contentration of Nattokinse and study the pharmacokinetic parameters of it in rabbits after intravenous administration. ELISA was set up by making the antiserum,then purificating the antibody from antiserum of Nattokinse and marking it with peroxidase. NK was administered by intravenous injection at the dose of 0.00707g/kg ?b?w in rabbits, and bleed at 5min,10min,20min,40min,60min,75min,120min,200min,270min,330min,420min and 510min after administration, then measure the contentration of drug in serum. The results indicated that ELISA had a high speciality, repeatability and seneibility; the drug concentration-time curve of Nattokinse in rabbit gotten by experiment was conform to two compartment opening model without absorption. The main pharmacokinetic parameters of NK as follows, Pistribution half-life T1/2α(min) is 21.36±2.081, Elimination half-life T1/2β(min) is 108.9±8.265, apparent volume of distribution Vd(L) is 0.255±0.022, V1(L) is 0.089±0.005, clearance CL(L/min) is 0.0018±0.0004, the area under curve AUC0-510 (mg/L*min) is 4300.5±185.2；AUC0-∞ (mg/L*min) is 4413.2±182.62.