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

doi:10.12140/j.issn.1000-7423.2022.02.004

Abstract

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

CLC Number:

R384.113

ZHANG Xia, YAN Feng, MU Xiao-hui, LIN Zi-min, NIE Ying, CHENG Jin-zhi, SHANG Zheng-ling, WU Jia-hong, , , . 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.

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蛋白浓度/nmol·L^-1 Protein concentration/ nmol·L^-1	聚集率/%Aggregation rate/%	聚集抑制率/% Aggregation inhibition rate/%
2 083.30	42.67 ± 6.65^a	35.75 ± 14.72^a
416.67	39.40 ± 3.10^a	40.47 ± 4.67^a
83.33	56.52 ± 1.61^a	14.88 ± 2.41^a
16.67	32.59 ± 5.37^a	54.21 ± 7.55^a
3.33	28.99 ± 8.47^a	59.27 ± 11.90^a
0.67	64.68 ± 1.86	9.12 ± 2.61
0.13	62.27 ± 8.47	12.51 ± 11.90
0.02	69.32 ± 1.71	2.60 ± 2.37
阴性对照 Negative control	69.26 ± 4.48	0

检测指标 Detection index	样品 Sample	不同浓度rAael-yellow-c蛋白 rAael-yellow-c protein with different concentration			阴性对照 Negative control
检测指标 Detection index	样品 Sample	0.12 μmol/L	1.20 μmol/L	12.00 μmol/L	阴性对照 Negative control
PT	1	13.63 ± 0.74	14.16 ± 1.38	15.03 ± 1.40	14.30 ± 0.46
	2	13.73 ± 0.64	14.38 ± 1.09	16.01 ± 2.28	14.44 ± 1.39
	3	14.10 ± 0.181	13.32 ± 0.87	13.55 ± 0.39	13.96 ± 0.12
APTT	1	32.76 ± 1.82	31.94 ± 0.77	30.33 ± 1.51	33.34 ± 0.79
	2	38.46 ± 4.23	41.78 ± 3.88	44.01 ± 6.47	38.40 ± 2.60
	3	37.59 ± 0.33	37.67 ± 1.78	38.52 ± 1.49	39.29 ± 1.91
TT	1	21.15 ± 0.78	21.05 ± 0.65	24.37 ± 2.04	22.90 ± 0.78
	2	21.20 ± 0.51	23.12 ± 2.83	25.30 ± 2.29	21.60 ± 2.63
	3	19.12 ± 0.45	19.70 ± 1.28	21.85 ± 1.38	18.56 ± 1.24