埃及伊蚊黄蛋白c基因的克隆、表达及其体外抗凝血作用

doi:10.12140/j.issn.1000-7423.2022.02.004

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (2): 153-158.doi: 10.12140/j.issn.1000-7423.2022.02.004

埃及伊蚊黄蛋白c基因的克隆、表达及其体外抗凝血作用

张霞¹(), 颜凤³, 牟小会¹, 林紫敏¹, 聂映¹^,^*(), 程金芝¹, 商正玲⁴, 吴家红¹

1.贵州医科大学寄生虫学教研室,贵安 550025
2.贵州医科大学基础医学院现代病原生物学特色重点实验室,贵安 550025
3.遵义医科大学第三附属医院（遵义市第一人民医院）检验科,遵义 563099
4.贵州医科大学免疫学教研室,贵安 550025

收稿日期:2021-09-29 修回日期:2021-11-08 出版日期:2022-04-30 发布日期:2022-04-12
通讯作者: 聂映,吴家红
作者简介:张霞（1996-）,女,硕士研究生,从事病媒生物防控与虫媒病方面的研究。E-mail： 2102924940@qq.com
基金资助:
国家自然科学基金面上项目(8197081668)

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

ZHANG Xia¹(), YAN Feng, MU Xiao-hui³, LIN Zi-min¹, NIE Ying, CHENG Jin-zhi¹, SHANG Zheng-ling, WU Jia-hong¹^,^*(), 2, 2, 2^*

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:2021-09-29 Revised:2021-11-08 Online:2022-04-30 Published:2022-04-12
Contact: WU Jia-hong, 2
Supported by:
National Natural Science Foundation of China(8197081668)

摘要/Abstract

摘要：

目的克隆表达埃及伊蚊黄蛋白c（Aael-yellow-c）基因,并探讨rAael-yellow-c蛋白体外抗凝血作用。方法 RT-PCR扩增Aael-yellow-c成熟肽基因,纯化后的片段与pEASY-E1载体连接,转化至大肠埃希菌DH5α感受态细胞,取菌液进行PCR、双酶切和测序鉴定。0.1 mol/L异丙基-β-D-硫代半乳糖苷诱导蛋白表达后,镍柱亲和层析纯化重组蛋白,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Western blotting）分析重组蛋白表达情况。比浊法观察不同浓度rAael-yellow-c蛋白对二磷酸腺苷诱导的血小板聚集活性影响。手工法检测不同浓度rAael-yellow-c蛋白对人血浆凝血酶原时间（PT）、部分凝血活酶时间（APTT）及凝血酶时间（TT）的影响。采用SPSS 18.0统计软件进行统计学分析,组间差异比较采用t检验。结果 Aael-yellow-c基因CDS区为1 287 bp,含27个氨基酸组成的信号肽,成熟肽序列长1 200 bp,编码399个氨基酸。RT-PCR扩增获得Aael-yellow-c基因片段,大小约为1 200 bp。菌液PCR、双酶切和测序鉴定显示,pEASY-E1-Aael-yellow-c质粒构建成功。SDS-PAGE结果显示,重组蛋白以包涵体形式表达;镍柱亲和层析纯化重组蛋白后获得单一条带。Western blotting结果显示,该蛋白可被His-tag抗体和抗rAael-yellow-c蛋白的兔血清多克隆抗体识别。血小板聚集抑制实验结果显示,2 083.30、416.66、83.33、16.67和3.33 nmol/L rAael-yellow-c蛋白可抑制由ADP诱导的血小板聚集,差异具有统计学意义（t = 7.09、10.39、5.39、10.54和8.93,P < 0.01）,其中3.33 nmol/L的重组蛋白对ADP诱导的血小板聚集抑制作用最强,抑制率为（59.27 ± 11.90）%;0.67、0.13和0.02 nmol/L的重组蛋白对ADP诱导的血小板聚集作用无影响（t = 2.10、1.33和0.00,P > 0.05）。内外源凝血实验结果显示,与阴性对照组相比,0.12、1.20 和12.00 μmol/L rAael-yellow-c分别作用于人血浆时,PT分别为13.63~14.10、13.32~14.38和13.55~16.01 s,差异无统计学意义（t = 1.33、0.63和1.00,P > 0.05）;APTT分别为32.76~38.46、31.94~41.78和33.34~39.29 s,差异无统计学意义（t = 0.47、0.06和0.24,P > 0.05）;TT分别为19.12~21.20、19.7~23.12和21.85~25.30 s,差异无统计学意义（t = 0.47、0.24和1.60,P > 0.05）。结论获得的rAael-yellow-c蛋白主要通过抑制ADP诱导的血小板聚集作用帮助蚊虫吸血。

关键词: 埃及伊蚊, yellow-c基因, 血小板聚集, 抗凝血

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

中图分类号:

R384.113

张霞, 颜凤, 牟小会, 林紫敏, 聂映, 程金芝, 商正玲, 吴家红. 埃及伊蚊黄蛋白c基因的克隆、表达及其体外抗凝血作用[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 153-158.

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

埃及伊蚊黄蛋白c基因的克隆、表达及其体外抗凝血作用

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

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