Prokaryotic expression and anticoagulant activity of Boophilin-H2,a protease inhibitor of Rhipicephalus Linnaei Kunitz type
Objective To clone Rhipicephalus linnaei Boophilin-H2 gene,construct the recombinant expression vector,express the Boophilin-H2 recombinant protein in Escherichia coli,and assess its anticoagulant activity.Methods Specific primers were designed to amplify the Boophilin-H2 gene fragment using cDNA,synthesized from engorged Rhipicephalus linnaei tick RNA through reverse transcription,as a template.The gene fragment was cloned and connected to plasmid pSmart-I,and the recombinant expression vector pSmart-I/Boophilin-H2 was constructed.The recombinant expression vector was verified by double restriction enzyme digestion with Bam HⅠand XhoⅠ,transferred into the competent state of Escherichia coli BL21(DE3,and expressed under low-temperature induction with IPTG.The recombinant protein was purified by Ni-NTA Resin,and its expression and purification were detected by 12.5%SDS-PAGE.The femoral venous blood of New Zealand white rabbits was collected by 3.8%sodium citrate blood collection tube,and the upper plasma was centrifugally separated to measure the anticoagulant activity of the recombinant protein using four test plates of in vitro coagulation.Results A 387 bp gene fragment of Boophilin-H2 of Rhipicephalus linnaei was successfully amplified and cloned;the prokaryotic expression vector pSmart-I/Boophilin-H2 was constructed and verified by double enzyme digestion.Following induction with 0.8 mmol/L IPTG for 16 hours in Escherichia coli,SDS-PAGE showed that the recombinant protein was expressed in the supernatant primarily in a soluble form,with the Boophilin-H2 recombinant protein approximately 35 000 in size.The anticoagulant activity assays of the purified recombinant protein Boophilin-H2 showed that the recombinant protein significantly prolongs the activated partial thromboplastin time(APTT)in a concentration-dependent manner,while its effects on thrombin time(TT),prothrombin time(PT),and fibrinogen(FIB)levels were not significant.Conclusions The expression vector for Boophilin-H2 was successfully constructed,and its product exhibited potent APTT anticoagulant activity,implying its role in the intrinsic coagulation pathway,possibly acting upon intrinsic coagulation factors VIII,XI,and XII to inhibit blood coagulation.This study provides a reference and theoretical foundation for further research and development of tick control vaccines and anticoagulant drugs.
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