中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
Chinese Journal of Immunology
2015年
11期
1505-1509
,共5页
颜秋霞%陈彩蓉%李介华%周秀琴%冼英杰%陈润强%蔡志明%唐爱发
顏鞦霞%陳綵蓉%李介華%週秀琴%冼英傑%陳潤彊%蔡誌明%唐愛髮
안추하%진채용%리개화%주수금%승영걸%진윤강%채지명%당애발
人睾丸特异性新基因%合成肽%单克隆抗体
人睪汍特異性新基因%閤成肽%單剋隆抗體
인고환특이성신기인%합성태%단극륭항체
Human testis-specific conserved gene%Synthesized peptide%Monoclonal antibody
目的:制备和初步鉴定人睾丸特异性新基因hTSC29( GenBank 登录号: BC032957)合成肽的单克隆抗体( mAb)。方法:利用生物信息学方法对hTSC29的蛋白质序列进行分析和预测,根据免疫原性、表露性、亲疏水性及柔韧性4个参数,合成一段由18个氨基酸组成的多肽,并以此为抗原免疫BALB/c小鼠,取其脾细胞和小鼠骨髓瘤细胞融合后,通过有限稀释法筛选阳性克隆,采用ELISA法、Western blot、免疫组化和免疫荧光法鉴定hTSC29 mAb的特异性。结果:获得了1株稳定分泌hTSC29 mAb的杂交瘤细胞株,分泌的抗体属IgG2b(κ)亚型,杂交瘤细胞培养上清效价达到1∶104;Western blot鉴定表明,制备hTSC29 mAb可特异性地识别人睾丸总蛋白中Mr约为60000处的蛋白;免疫组织化学和免疫荧光结果显示hTSC29蛋白主要定位在正常人睾丸组织中的精母细胞和精子细胞。结论:成功建立了1株稳定分泌抗hTSC29 mAb的杂交瘤细胞株,其分泌的抗体效价高、特异性强,为进一步研究hTSC29的功能提供了特异的检测工具。
目的:製備和初步鑒定人睪汍特異性新基因hTSC29( GenBank 登錄號: BC032957)閤成肽的單剋隆抗體( mAb)。方法:利用生物信息學方法對hTSC29的蛋白質序列進行分析和預測,根據免疫原性、錶露性、親疏水性及柔韌性4箇參數,閤成一段由18箇氨基痠組成的多肽,併以此為抗原免疫BALB/c小鼠,取其脾細胞和小鼠骨髓瘤細胞融閤後,通過有限稀釋法篩選暘性剋隆,採用ELISA法、Western blot、免疫組化和免疫熒光法鑒定hTSC29 mAb的特異性。結果:穫得瞭1株穩定分泌hTSC29 mAb的雜交瘤細胞株,分泌的抗體屬IgG2b(κ)亞型,雜交瘤細胞培養上清效價達到1∶104;Western blot鑒定錶明,製備hTSC29 mAb可特異性地識彆人睪汍總蛋白中Mr約為60000處的蛋白;免疫組織化學和免疫熒光結果顯示hTSC29蛋白主要定位在正常人睪汍組織中的精母細胞和精子細胞。結論:成功建立瞭1株穩定分泌抗hTSC29 mAb的雜交瘤細胞株,其分泌的抗體效價高、特異性彊,為進一步研究hTSC29的功能提供瞭特異的檢測工具。
목적:제비화초보감정인고환특이성신기인hTSC29( GenBank 등록호: BC032957)합성태적단극륭항체( mAb)。방법:이용생물신식학방법대hTSC29적단백질서렬진행분석화예측,근거면역원성、표로성、친소수성급유인성4개삼수,합성일단유18개안기산조성적다태,병이차위항원면역BALB/c소서,취기비세포화소서골수류세포융합후,통과유한희석법사선양성극륭,채용ELISA법、Western blot、면역조화화면역형광법감정hTSC29 mAb적특이성。결과:획득료1주은정분비hTSC29 mAb적잡교류세포주,분비적항체속IgG2b(κ)아형,잡교류세포배양상청효개체도1∶104;Western blot감정표명,제비hTSC29 mAb가특이성지식별인고환총단백중Mr약위60000처적단백;면역조직화학화면역형광결과현시hTSC29단백주요정위재정상인고환조직중적정모세포화정자세포。결론:성공건립료1주은정분비항hTSC29 mAb적잡교류세포주,기분비적항체효개고、특이성강,위진일보연구hTSC29적공능제공료특이적검측공구。
Objective:To prepare monoclonal antibody ( mAb ) against human testis-specific conserved gene ( hTSC29 ) peptides and characterize its immunological and biological features.Methods:According to bioinformatics analysis and prediction of the antigenicity, surface property, hydrophilicity and flexibility of hTSC29, a 18-amino acid residue partial peptide of hTSC29 was synthesized,then immunized the BALB/c mice for preparing antiserum.The mAb against hTSC29 was produced using the routine hybridoma technique.The properties of the mAb against hTSC29 were identified by ELISA, Western blot and immunohistochemistry staining.Results:After cell fusion and subcloning, one hybridoma cell lines secreting specific mAb against hTSC29 protein were obtaind.The Ig subclass of the mAb was IgG2b(κ).ELISA detection showed that the titer of mAbs in cultured was 1∶104.Western blot analysis proved that the mAb could specifically recognize Mr 60 000 protein in human testis total protein.The hTSC29 protein main located at circumference of spermatocyte and spermatid in human testis tissue by immunohistochemistry staining and immunofluorescence assay.Conclusion:One hybridoma cell lines which can secrete specific mAb against hTSC29 protein with high titers and specificity have been established successfully.The mAb will provide efficient tools for functional studies of hTSC29 expressed in spermatogenesis.