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

作　　者：张霞 颜凤 牟小会 林紫敏 聂映 程金芝[1,2] 商正玲 吴家红[1,2] ZHANG Xia;YAN Feng;MU Xiao-hui;LIN Zi-min;NIE Ying;CHENG Jin-zhi;SHANG Zheng-ling;WU Jia-hong(Department of Parasitology,Guizhou Medical University,Gui’an 550025,China;Key Laboratory of Modern Pathogenic Biology,School of Basic Medicine,Guizhou Medical University,Gui’an 550025,China;Clinical Laboratory,the Third Affiliated Hospital of Zunyi Medical University,the First People’s Hospital of Zunyi,Zunyi 563099,China;Department of Immunology,Guizhou Medical University,Gui’an 550025,China)

机构地区：[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

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第2期153-158,167,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金面上项目(8197081668)。

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1287 bp,含27个氨基酸组成的信号肽,成熟肽序列长1200 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1200 bp。菌液PCR、双酶切和测序鉴定显示,pEASYE1-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,2083.30、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12.00μmol/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�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 p EASY-E1,which was transformed into Escherichia coli DH5αcompetent 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 1200 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 12.00μmol/L)treated human plasma had PT of 13.63-14.10 s,13.3

关 键 词：埃及伊蚊 yellow-c基因 血小板聚集 抗凝血 

分 类 号：R384.113[医药卫生—医学寄生虫学]