The Initial Study Of The Recombinant Human Acetylcholinesterase Treatment Of Organophosphorus (Dichlorvos) Poisoning

Objective:The human total RNA was extracted from the normal fetal brain. We cloned acetylcholinesterase (hAChE) cDNA, constructed cloning expression vector pUCm-T/AChE and recombinant expression vector pET-AChE, recombinant and expression human acetylcholinesterase (hAChE), and obtained recombinant human acetylcholine esterase (rhAChE). And thus its molecular characteristics and biological characteristics were detected, then selected with high binding capacity of organic phosphorus and activity of certain target protein,for further rhAChE in vivo studies and clinical application of foundation,so as to open the way of the treatment of organophosphorus poisoning.Methods:According to the Gene Bank in the AChE gene sequences, we use Primer Primier5 software to design and synthesis specific primers that was used to gene amplification for hAChE. We added restriction site of EcoR I on the upstream site of 5'and Xhol I on the Downstream site of 3'. The human total RNA was extracted from the normal fetal brain. The hAChE cDNA fragment was amplified by RT-PCR with the RNA as a template. The purified PCR amplification products connectioned with the cloning vector of pUCm-T. Then it constructioned the cloning and expression vector, named the pUCm-T/AChE. After that, we transformed the vector into E. coli DH5αcompetent cells. The recombinant plasmid was induced to express in DH5αcompetent cells by IPTG, then we screened white colonies. The plasmid DNA was minipreped, and identified by restriction enzyme digestion and PCR, so it was sequenced and analysised. The pUCm-T/AChE cutted by restriction enzymes EcoR I and Xho I. AChE gene fragments recovered and digested by the pET-28a (+) expression vector.Then it constructioned the recombinant and expression plasmid, named the pET-AChE. The recombinant plasmid was transformed into E. coli BL21 and induced to express by IPTG. We filtered positive plasmids by Kan+, and identified the recombinant plasmid by PCR. The induction and no induction of whole cell engineering bacteria protein detectioned by SDS-PAGE electrophoresis, and then recombinant protein was purified and quantitatived analysis.According to the modified Ellman method (colorimetric), we detectioned normal blood acetylcholinesterase and rhAChE activity, and compared. In vitro, we directly mixed the rhAChE with different doses of organic phosphate (DDVP). According to the activity of rhAChE, we could indirectly get the binding activity of rhAChE and organic phosphorus.Results:The total RNA was measured by ultraviolet spectrophotometry, the result was that OD260/OD280> 1.8, OD260/OD270> 1.2, and showed that there was no residue in protein and phenol. We taken lul samples of RNA for formaldehyde denaturing gel electrophoresis, showed two bands of 28S and 18SRNA, indicaed that the basic integrity of RNA. After the agarose gel electrophoresis and enzyme digestion, coding sequence of hAChE was 1845bp, consistented with the GenBank DNA database of human acetylcholinesterase gene sequence. We successfully constructed cloning and recombinant expression vectors about pUCm-T/AChE and pET-AChE. Its sequence is consistent with the reported sequence in the GenBank. SDS-PAGE and Western blot analysis showed that rhAChE molecular weight is 68.2kDa, the concentration is 0.75mg/ml. Most of recombinant hAChE protein expressed in the form of inclusion bodies which found in the deposit of cell lysates. Normal human whole blood and plasma cholinesterase activity were 4.68±0.24,0.48±0.12 ( x±s); 0.75mg/ml rhAChE10μl, 50μl, 100μl of the activities were 0.16±0.05,0.87±0.05, 1.27±0.10 ( x±s); the restructuring hAChE has even higher activity than normal plasma ChE.Conclusion:The cDNA fragment of hAChE was successfully cloned by RT-PCR. We constructed cloning expression vector pUCm-T/AChE and recombinant expression vector pET-AChE, then the rhAChE was obtained. rhAChE have some biological activities, and binding activity with organic phosphorus pesticides, and even higher activity than normal plasma ChE. It could partially replace human AChE. The angle of the enzymatic treatment has opened up new ways of organophosphate poisoning.