钩端螺旋体澳洲群体特异性McAb的建立,鉴定及群特异性...

Spleen cells of BALB/c mice immunized with whole Leptospira interrogans serogroup Australis serovar australis strain 620 were fused with myeloma cells line SP2/0. Specificities of four McAbs determined by MAT. 2E1 McAb (IgG3) reacted with 11 serovars, of the Australis serogroup, but did not react with 22 representative serovars of L. interrogans in 20 serogroups, L. biflexa strain patoc I and Leptonema illini strain 3055. 2E1 McAb showed serogroup specificity for Australis by agglutination and the other 3 McAbs showed partial serogroup specificity. We compared the outer envelope (OE) protein profiles of serovar australis strain 620 with those of two pathogenic L. interrogans serovar lai strain 601 and serovar hebdomadis strain 156 by SDS-PAGE. 63kd protein profile was only found in the OE of strain 620, and the quantity of 42kd protein of strain 620 was greater than that of strain 601 and 156. The immunoblotting revealed that 2E1 McAb reacted with a 34kd band in the OE preparation of serovar australis strain 620, but did not react with that of other two L. interrogans. 2E1 McAb also did not react with OE of non-pathogenic leptospires. It was suggested that 34kd protein might contain the antigenic determinants which were shared by leptospires of Australis serogroup.     

保护性MbE_4B_7G_5对问号状钩体疏水外膜蛋白OmpL_(39)的免疫印迹分析

选用具有高效价、特异性的黄疸出血群保护性单克隆抗体（ＭｃＡｂ）Ｅ４Ｂ７Ｇ５，用Ｗｅｓｔｅｒｎ－ｂｌｏｔｉｎｇ，对ＴｒｉｔｏｎＸ－１１４（ＴＸ－１１４）抽提的６株问号状钩体（０１７、６０１、６０３、６０９、６２０、２４５株）的疏水外膜蛋白进行分析鉴定。结果表明，提纯的０１７、６０１、６０３、６０９株问号状钩体３９ｋｄ疏水外膜蛋白（ＯＭＰ）有明显的免疫反应，而６２０、２４５株此带区无明显的免疫识别。这一结果提示：用ＭｂＥ４Ｂ７Ｇ５筛选问号状钩体保护性抗原，分离、纯化３９ｋｄ的疏水外膜蛋白（ＯｍｐＬ３９）在构建钩体基因工程疫苗中有重要的研究价值。     

保护性Mb E4B7G5对问号状钩体疏水外膜蛋白OmPL39的免疫…

选用具有高效价、特异性的黄疸出血群保护性单克隆抗体（ＭｃＡｂ）Ｅ４Ｂ７Ｇ５，用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ，对Ｔｒｉｔｏｎ Ｘ－１４４（ＴＸ－１４４）抽提的６株问号状钩体（０１７、６０１、６０３、６０９株）的疏水外膜蛋白进行分析鉴定。结果表明，提纯的０１７、６０１、６０３、６０９株问号状钩体３９ｋｇ疏水外膜蛋白（ＯＭＰ）有明显的免疫反应，而６２０、２４５株此带区无明显的免疫识别。这一结果提示：用     

五株钩体膜蛋白和脂多糖的SDS—PAGE,ZD—PAGE图谱的比较研究

We applied SDS-PAGE, 2D-PAGE and Western blot to analyse the outer envelopes protein and LPS of five strains of leptospires. The work would lay foundations for taxonomy, the development of vaccination regimens and the elucidation of pathogenic mechanisms. The outer envelope proteins of leptospires were analyzed by SDS-PAGE and silver staining. We found that the protein profiles of the pathogenic leptospires were basically identical. A comparison of the protein profiles of the pathogenic L. with those exhibited by two nonpathogenic L. indicated that there was no obvious relationship between these organisms and any of the L. interrogans strains examined. The quantity of 21.5 kd protein of strain 017 was greater than that of strain 601 and 156. Approximately 200, 225, 238 distinct polypeptides were detected in the strain 017, 601 and 156 in 2D-PAGE by silver staining respectively. The profiles of 2D-PAGe showed obvious differences in pI. The pI of strain 017, 601 and 156 were mainly 6.68-7.4, 6.55-6.9, 5.85-7.1 respectively. The 21.5 kd protein of strain 017 was made up of six polypeptides. Our immunoblots revealed that McAb (LB1) reacted with a 41 kd antigen, which was common to the three virulent leptospires tested. SDS-PAGE profiles of silver stained outer envelope LPS of pathogenic L. differed greatly from those of the nonpathogenic L. There was a distinct differences between strain Patoc I and 3055. Our studies showed that each of the five strains of leptospires possessed characteristic outer envelope LPS, which may be used to identify the genus, species and serovars of a strain of L.     

钩端螺旋体澳洲群群特异性McAb的建立、鉴定及群特异性抗原检测

作者成功地获得4株分泌抗澳洲群钩瑞螺旋体的单克隆抗体(McAb)杂交瘤。其中2E_1株能与所选澳洲群的11个型的钩体都发生凝集反应,而不与澳洲群外的20群22个型的钩体、非致病性Patot Ⅰ株钩体和伊利尼细螺旋体3055株发生凝集反应。由此表明,2E_1 McAb具有澳洲群钩体的群特异性。用SDS-PAGE、免疫印迹法对澳洲型620株、赖型601株、七日热型156株、Patoc Ⅰ株及3055株外膜蛋白中McAb识别的抗原成分进行检测,结果2E_1 McAb能特异地识别澳洲型620株34kd外膜蛋白区带。其余3株McAb具有澳洲群钩体部分群特异性。     

针对问号钩体赖型外膜的抗原分析

作者采用SDS-聚丙烯酰胺凝胶电泳和免疫印迹技术,对黄疸出血群赖型017株和601株、七日热群七日热型156株、澳洲群澳洲型620株以及塞马伦群帕托克型Patoc Ⅰ株等5株钩体外膜进行分析。检测到具有免疫保护作用和抑制钩体粘附作用的单克隆抗体E_4B_7D_5能特异地识别赖型017株和601株钩体外膜的34.5kd和39.5kd两条蛋白带,而同其余3株钩体外膜蛋白无反应。由此提示该两条蛋白带可能为钩体外膜保护性抗原。     

应用胶体金技术对钩体抗原超微定位的研究

The rabbit anti-mouse antibodies were successfully labelled with colloidal gold. The distribution of leptospiral antigens in ultrastructure was researched with McAbs and labelled antibodies. It was found that colloidal gold particles were mainly distributed at the outer membrane of leptospiral strain O17 cells, which indicated that the antigens recognized by McAbs 1A7E7, and 2F9D4 were localized at the outer membrane of leptospiral cells. It was thought that colloidal technique would provide a method for research on the antigen distribution of leptospiral cells in ultrastructure.     

Observation of monoclonal antibody E4B7D5 inhibitory effect on leptospiral adherence using scanning electron microscope]

BALB/c mice were immunized intraperitoneally with outer envelopes of serogroup icterohaemorrhagiae lai serovar strain 017 leptospires. Monoclonal antibody (McAb) E4B7D5 against outer envelopes (IgG1, agglutinating titre 1:25,600) was produced by hybridoma technique. Passive immunoprotection experiments have demonstrated the immunoprotection of McAb E4B7D5 against strain 017 leptospires. Effect of McAb E4B7D5 on leptospiral adherence to the surface of normal human pulmonary embryonic fibroblasts was observed by using scanning electron microscope. The results indicated that the leptospiral adherence noted in various agglutinating titre McAb E4B7D5 groups was less frequent than that in the three control groups. It was concluded that the inhibitory effect of McAb E4B7D5 on leptospiral adherence may play a role in the immunoprotection.     

赖型017株钩体疏水外膜蛋白及其免疫特征的研究

采用低浓度非离子型去污剂ＴｒｉｔｏｎＸ－１１４（ＴＸ－１１４）后提和分离问号状钩端螺旋体（钩体）黄疸出血群赖型０１７株（０１７），选择性地将钩体外膜蛋白分离为ＴＸ－１１４疏水相和亲水相两个部分。疏水相外膜蛋白含５条主要蛋白区带，其分子量分别为６６ｋｄ、３９ｋｄ、３５ｋｄ、２７ｋｄ和６ｋｄ，，ａｄｋｈｗｃｙ ３９ｋｄ外膜蛋白区带可特异地分别被抗全０１７血清，抗０１７外膜血清和抗０１７保护性单克隆抗体     

抗旋毛形线虫单克隆抗体的制备和抗原定位研究

BALB/c mice were infected per os with the infective larvae (L1) of Trichinella spiralis, and challenged by injecting 0.2ml soluble L1 antigen 3 days before cell fusion. SP2/0 myeloma and immune spleen cells were fused at the ratio of 1:5 in the presence 30%PEG (MW 4,000). The fusion rate and antibody positive rate were 92.1% and 16.6% respectively using ELISA. Among these the chromosomes of 4 strains were 2n greater than 100 and that of the SP2/0 myeloma cells 2n less than or equal to 70. The titer of McAb in the ascites was found to be 1/640 or 1/1,280. Eight strains were of IgG1, 1 IgG2a and 3 IgM in an agar system. Three strains of McAbs, which could recognize specifically the L1 antigen fractions by immuno-blotting technique, were used as probes to localize the target antigens in sections of T. spiralis L1. Staining was performed using an indirect technique consisting of goat anti-mouse IgG-conjugated horseradish peroxidase on de-paraffin sections, and substrate DAB. The results revealed that the antigens reacted most strongly with the specific McAbs were located in the cuticle and stichosome, followed by the lining of gut. Among the 3 strains of McAbs used, 2E5F11A5 reacted most strongly with the antigen, followed by 2E5E9E4. 2E5E9G5 was the weakest.