利什曼原虫K26基因的克隆、表达及用于我国内脏利什曼病特异性抗体检测的效果评价  

Cloning and expression of the K26 gene of Leishmania and evaluation of detection of specific antibodies against visceral leishmaniasis in China

作　　者：丁丹 王颖 高春花 莫筱瑾 石锋 张璟 贾孝凯 危芙蓉 DING Dan;WANG Ying;GAO Chun-hua;MO Xiao-jin;SHI Feng;ZHANG Jing;JIA Xiao-kai;WEI Fu-rong(Center for Disease Control&Prevention,Pudong New Area,Shanghai 200136,China;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research).NHC Key Laboratory of Parasite and Vector Biology.WHO Collaborating Centre for Tropical Diseases,National Center for International Research on Tropical Diseases,Shanghai 200025,China)

机构地区：[1]上海市浦东新区疾病预防控制中心,上海200136 [2]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心),国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,国家级热带病国际联合研究中心,上海200025

出　　处：《中国人兽共患病学报》2024年第8期763-767,共5页Chinese Journal of Zoonoses

摘　　要：目的克隆并表达分离于我国3种类型的利什曼原虫K26基因,并用其检测我国内脏利什曼病特异性抗体,同时进行效果评价。方法分别将我国新疆喀什(KS-6株)、四川九寨沟县(SC6株)和新疆伽师县(JIASHI-1株)K26基因进行全基因合成并加入Bam H I与Xho I酶切位点,分别将K26基因连接至双酶切的pET32a表达载体,转入大肠埃希菌(E.coli)BL21(DE3)中经1 mmol/L的异丙基-β-D-半乳糖苷(IPTG)进行诱导表达,用组氨酸标签亲和纯化柱(Ni-NTA树脂)纯化重组蛋白。用制备的3种抗原分别作为包被抗原,以酶联免疫吸附试验(ELISA)检测病原学确诊的内脏利什曼病患者及其它寄生虫病患者和健康者的血清中和抗体,以评价其检测的敏感性和特异性。用美国InBios公司rK39试条进行平行检测,比较3种抗原检测的敏感性。结果成功构建利什曼原虫pET32a-K26重组质粒,并在原核细胞中成功表达。以KS-6株、SC6株、JIASHI-1株利什曼原虫重组K26蛋白为包被抗原的ELISA法和rK39试条法检测黑热病患者血清的敏感性分别为90.00%(99/110)、92.73%(102/110)、90.91%(100/110)和93.64%(103/110),共检测45份其他寄生虫病患者(包括日本血吸虫病、疟疾、细粒棘球蚴病、华支睾吸虫病、卫氏并殖吸虫病和弓形虫病)的血清均无交叉反应,健康者血清(40份)也无假阳性反应,特异性均为100.00%。KS-6株、SC6株和JIASHI-1株利什曼原虫重组K26蛋白ELISA法和rK39试条法的阳性检出率差异无统计学意义(χ^(2)_(KS)-6=0.97,P=0.33;χ^(2)_(SC6)=0.07,P=0.79;χ^(2)_(JIASHI)-1=0.57,P=0.45)。3种K26抗原的阳性检出率差异无统计学意义(χ^(2)=0.53,P=0.97)。结论重组K26抗原在内脏利什曼病诊断上具有潜在的应用价值。To clone and express the K26 gene of Leishmania isolated from three types of visceral leishmaniasis epidemic areas in China and evaluate its effect on detecting specific antibodies against visceral leishmaniasis.The K26 fragments from Leishmania isolated KS-6,SC6 and JIASHI-1 was synthesized and cloned into pET32a vector.The recombinant plasmid pET32a-K26 was transformed into Escherichia coli BL21 strains and induced by isopropyl-β-D-thiogalactopyranoside(IPTG).The expressed recombinant protein was purified by the His-tag ged affinity column(Ni-NTA).Serum samples of 110 visceral leishmaniasis patients were used for evaluating the sensitivity by ELISA.Serum samples from patients with malaria,schistosomiasis japonica,cystechinococcosis,toxoplasmosis,paragonimiasis,clonorchiosis and 40 healthy people were used for evaluating the specificity.Detection results of ELISA were compared with that of rK39 strip of American InBios company.Comparation among three K26 antigens were given by χ^(2) test.The sensitivity of the recombinant K26 protein of KS-6,SC6 and JIASHI-5 strains of Leishmania and rK39 strip test to detect the sera of patients with visceral leishmaniasis was 90.00%(99/110),92.73%(102/110),90.91%(100/110)and 93.64%(103/110),respectively.There was no cross reactivity with malaria(10),schistosomiasis japonica(10),cystechinococcosis(10),toxoplasmosis(5),paragonimiasis(5)and clonorchiosis(5),and 40 sera from healthy people were also negative.The specificity was 100.00%.There was no statistical difference in the sensitivity of the recombinant K26 protein of KS-6,SC6 and JIASHI-1 strains of Leishmania and rK39 strip test,χ^(2) values are 0.97,0.07 and 0.57 respectively and the P values are 0.33,0.79 and 0.45,respectively.There was no statistical difference in the sensitivity of three K26 antigens(χ^(2)=0.53,P=0.97).Conclusion The recombinant K26 antigen has potential application value in the diagnosis of visceral leishmaniasis.

关 键 词：内脏利什曼病 rK26 基因克隆 表达 酶联免疫吸附试验 rK39 评价 

分 类 号：R382.2[医药卫生—医学寄生虫学]