Prokaryotic expression and anticoagulant activity of Boophilin-H2, a protease inhibitor of Rhipicephalus Linnaei Kunitz type
- Author:
ZHAO Peizhen
;
LI Yao
;
YU Lingling
- Publication Type:Journal Article
- Keywords:
Rhipicephalus Linnaei;
Kunitz protease inhibitor;
prokaryotic expression;
anticoagulant activity
- From:
China Tropical Medicine
2024;24(9):1106-
- CountryChina
- Language:Chinese
-
Abstract:
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 BamHⅠand 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.
- Full text:202511111547246991915.Prokaryotic expression and anticoagulant activity of Boophilin-H2, a protease inhibitor of Rhipicephalus Linnaei Kunitz type.pdf
