CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >

2022 , Vol. 40 >Issue 2: 153 - 158

DOI: https://doi.org/10.12140/j.issn.1000-7423.2022.02.004

ORIGINAL ARTICLES

Cloning and expression of yellow c gene from Aedes aegypti and its in vitro action of anti-coagulation

  • ZHANG Xia ,
  • YAN Feng ,
  • MU Xiao-hui ,
  • LIN Zi-min ,
  • NIE Ying ,
  • CHENG Jin-zhi ,
  • SHANG Zheng-ling ,
  • WU Jia-hong ,
  • 2 ,
  • 2 ,
  • 2
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  • 1. Department of Parasitology, Guizhou Medical University, Gui’an 550025, China
    2. Key Laboratory of Modern Pathogenic Biology, School of Basic Medicine, Guizhou Medical University, Gui’an 550025, China
    3. Clinical Laboratory, the Third Affiliated Hospital of Zunyi Medical University, the First People’s Hospital of Zunyi, Zunyi 563099, China
    4. Department of Immunology, Guizhou Medical University, Gui’an 550025, China

Received date: 2021-09-29

  Revised date: 2021-11-08

  Online published: 2022-04-12

Supported by

National Natural Science Foundation of China(8197081668)

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Abstract

Objective To clone and express the Aedes aegypti yellow-c(Aael-yellow-c) gene and investigate the in vitro anticoagulation activity of recombinant Aael-yellow-c protein. Methods The Aael-yellow-c mature peptide gene was amplified by RT-PCR, and the purified target gene segment obtained was connected to the plasmid pEASY-E1, which was transformed into Escherichia coli DH5α competent cell. The clones were verified with PCR, double restriction enzymes and sequencing. After induction with 0.1 mol/L IPTG, the recombinant protein was purified by Ni-affinity chromatography and identified by SDS-PAGE and Western blotting. The effect of rAael-yellow-c protein on human platelet aggregation induced by adenosine diphosphate (ADP) was observed by turbidimetry. The affect of rAael-yellow-c at different concentrations on prothrombin time (PT), activated thromboplastin time (APTT) and thrombin time (TT) of human blood plasma was detected manually. Results A 1 200 bp fragment of Aael-yellow-c gene was obtained by RT-PCR, and an pEASY-E1-Aael-yellow-c plasmid was successfully constructed by PCR, double restriction enzymes and sequencing. The recombinant protein was expressed in the form of inclusion body confirmed by SDS-PAGE and Western blotting, the purified recombinant protein was obtained by Ni-NTA. The results of the platelet aggregation inhibition experiment showed that 2083.30, 416.66, 83.33, 16.67 and 3.33 nmol/L rAael-yellow-c protein could inhibit platelet aggregation induced by ADP, and the difference was statistically significant(t = 7.09, 10.39, 5.39, 10.54 and 8.93, P < 0.01). The strongest inhibition effect was at 3.33 nmol/L, and the inhibitory rate was (59.27 ± 11.90)%; The recombinant proteins of 0.67, 0.13 and 0.02 nmol/L had no effect on ADP induced platelet aggregation, and the results were not statistically different (t = 2.10, 1.33 and 0.00, P > 0.05); The results the of internal and external source coagulation test showed that compared with the negative control group, rAael-yellow-c (0.12, 1.20 and 12.00 μmol/L) treated human plasma had PT of 13.63-14.10 s, 13.32-14.38 s and 13.55-16.01 s. The difference was not statistically significant (t = 1.33, 0.63 and 1.00, P > 0.05). APTT was 32.76-38.46 s, 31.94-41.78 s and 33.34-39.29 s, the difference was not statistically significant (t = 0.47, 0.06 and 0.24, P > 0.05). TT was 19.12-21.20 s, 19.7-23.12 s, 21.85-25.3 s, the difference was not statistically significant (t = 0.47, 0.24 and 1.60, P > 0.05). Conclusion The recombinant Aael-yellow-c protein obtained may be aidful for mosquito in blood sucking through suppressing platelet aggregation induced by ADP.

Key words： Aedes aegypti; Yellow c gene; Platlet aggregation; Anti-coagulation

Cite this article

ZHANG Xia , YAN Feng , MU Xiao-hui , LIN Zi-min , NIE Ying , CHENG Jin-zhi , SHANG Zheng-ling , WU Jia-hong , 2 , 2 , 2 . Cloning and expression of yellow c gene from Aedes aegypti and its in vitro action of anti-coagulation[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022 , 40(2) : 153 -158 . DOI: 10.12140/j.issn.1000-7423.2022.02.004

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