摘要
目的 筛选蓝氏贾第鞭毛虫（简称贾第虫，Giardia lamblia）α-8贾第素（α-8 giardin）的优势抗原表位肽，并分析其抗原性。 方法 采用DNAstar、Biosun软件及CLUSTAL W软件进行生物信息学分析，筛选出贾第虫α-8 giardin的3个优势抗原表位肽，即G1（7～17 aa）、G2（30～40 aa）和 G3（296～306 aa）；3个抗原表位肽分别与钥孔血蓝蛋白（KLH）偶联后（KLH-G1、KLH-G2、KLH-G3）免疫雌性青紫蓝家兔（每组2只），分别行背部皮下多点（8～10点）免疫注射，首次免疫各偶联蛋白0.5 mg/只（每点100 μl）。分别于首次免疫后14、21、28 d进行加强免疫（各偶联蛋白0.2 mg/只），于每次免疫前进行兔耳缘静脉采血，末次加强免疫后7 d颈动脉取血（各50 ml）。用间接ELISA检测3种（KLH-G1、KLH-G2和KLH-G3）免疫抗血清中IgG抗体效价；蛋白质印迹（Western blotting）分析3种免疫抗血清与重组蛋白rGiardin的抗原性。 结果 利用生物信息学相关软件筛选出的贾第虫α-8 giardin 3个候选抗原表位肽分别与KLH偶联后获得3个偶联蛋白（KLH-G1、KLH-G2、KLH-G3），经纯化后蛋白浓度分别为0.66、0.95和0.25 mg/ml。分别于加强免疫家兔后7 d，ELISA检测结果显示，3种（KLH-G1、KLH-G2、KLH-G3）免疫抗血清的抗体效价分别为1∶12800，1∶51200，1∶51200；SDS-PAGE分析结果显示，经纯化后的3种抗血清均在相对分子质量（Mr）约36 000处出现特异性的清晰条带，无其它杂带。Western blotting分析结果显示，3种抗血清均可特异性识别重组蛋白rGiardin。 结论 筛选出的贾第虫α-8 giardin 3个优势抗原表位肽与KLH偶联后免疫家兔，可诱导产生特异性IgG抗体；3种免疫抗血清中的特异性IgG抗体均可识别重组蛋白rGiardin；3个贾第虫α-8 giardin抗原表位肽均有较好的抗原性。
Objective To identify the dominant epitopes of α-8 giardin in Giardia lamblia, and analyze their immunoreactivity. Methods Three dominant epitopes G1（7-17 aa）, G2（30-40 aa） and G3（296-306 aa） were predicted for α-8 giardin by bioinformatic analysis using DNAstar, Biosun and CLUSTAL W softwares. Each epitope was conjugated to keyhole limpethemocyanin（KLH） and then used to immunize female Chinchilla rabbits via multi-point（8-10 points） subcutaneous injections on the back（0.5 mg conjugate/rabbit, 100 μl at each point）. Then the rabbits were boosted（0.2 mg conjugate/rabbit） on days 14, 21 and 28 after the first immunization. Blood was collected prior to each immunization, and carotid artery blood（50 ml） was collected on day 7 after the last immunization. The antibody titer for anti-KLH-G1, anti-KLH-G2 and anti-KLH-G3 sera was determined with indirect ELISA. The reactivity of the three anti-sera with recominant giardin（rGiardin） was analyzed with Western blotting. Results The concentration of the purified conjugates KLH-G1, KLH-G2, and KLH-G3 was 0.66, 0.95 and 0.25 mg/ml, respectively. ELISA showed that the antibody titer for anti-KLH-G1, anti-KLH-G2 and anti-KLH-G3 sera was 1∶12800, 1∶51200, 1∶51200, respectively. SDS-PAGE revealed a specific band at Mr 36 000 for the three anti-sera. Western blotting showed that the three anti-sera all specifically recognized rGiardin. Conclusion Immunization with the KLH-G1, KLH-G2, and KLH-G3 conjugates induced production of specific IgG antibody in rabbits, which can recognize rGiardin.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2016年第5期389-393,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.31360508)~~
