微小隐孢子虫甘油醛-3-磷酸脱氢酶的定位及活性检测  

ACTIVITY AND LOCALIZATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE(GAPDH)IN CRYPTOSPORIDIUM PARVUM

作　　者：邹贝贝 王东强[1] 尹继刚 朱冠 ZOU Bei-bei;WANG Dong-qiang;YIN Ji-gang;ZHU Guan(State Key Laboratory of Zoonoses Institute,Jilin University,Changchun 130062,Jilin,China;College of Veterinary Medicine,Jilin University,Changchun 130062,Jilin,China)

机构地区：[1]吉林大学人兽共患病研究所国家重点实验室,长春130062 [2]吉林大学动物医学学院,长春130062

出　　处：《寄生虫与医学昆虫学报》2023年第3期137-142,共6页Acta Parasitologica et Medica Entomologica Sinica

摘　　要：本研究以微小隐孢子虫甘油醛-3-磷酸脱氢酶(Cryptosporidium parvum glyceraldehyde-3-phosphate dehydrogenase,CpGAPDH)为对象,研究其亚细胞定位和酶动力学特征。设计特异性抗原多肽并免疫家兔,获得多克隆抗体,利用亲和纯化法获特异性抗体;经Western blot方法验证该抗体特异性,再通过间接免疫荧光法(IFA)进行蛋白定位。通过原核表达获得GST重组蛋白(GST-CpGAPDH),基于GAPDH的酶促反应利用辅酶NAD(H)或NADP(H)为电子受体或供体的原理,通过吸光度比色法监测反应中NAD(H)/NADP(H)的增加或减少鉴定其酶活性。结果显示,成功获得并纯化抗CpGAPDH抗体;Western blot分析显示该抗体可识别条带大小为40 kDa;IFA结果表明该蛋白主要分布于子孢子胞浆内,部分呈现颗粒状。利用重组蛋白确定了CpGAPDH的酶活性;在两种辅酶中,CpGAPDH更倾向于使用NAD(H)进行酶活反应。结果表明,本研究确定了CpGAPDH在虫体细胞的定位及酶活性检测,为进一步研究其生物学功能奠定了基础。To determine the subcellular localization and basic enzyme kinetics of Cryptosporidium parvum glyceraldehyde-3-phosphate dehydrogenase(CpGAPDH),one of the key enzymes in the glycolytic pathway.A CpGAPDH-specific short peptide was designed,synthesized and used to immunize two specific pathogen-free rabbits.The antisera were affinity-purified and used in Western blot analysis and indirect immunofluorescence(IFA).A GST-fusion recombinant protein(GST-CpGapDH)was expressed in bacteria and purified for determining basic enzyme kinetic data by spectrophotometry.Rabbit anti-CpGAPDH polyclonal antibodies were obtained and affinity-purified.Western blot analysis showed that the band size of the antibody was 40 kDa.IFA results showed that the protein was mainly distributed in the cytoplasm of sporozoites with some in granular form.Using recombinant GST-CpGAPDH,the basic enzyme parameters were determined.For coenzymes,CpGAPDH preferred using NAD(H)rather than NADP(H).The subcellular localization and enzyme activity detection of CpGAPDH was determined.This study serves as the basis to further study of the biological function and identification of selective inhibitors of CpGAPDH.

关 键 词：微小隐孢子虫 糖酵解途径 甘油醛-3-磷酸脱氢酶(GAPDH) 酶活动力学 蛋白定位 

分 类 号：R53[医药卫生—内科学]