CAJ | 학술논문

目的：筛选并鉴定致病性问号钩端螺旋体（简称钩体）外膜蛋白Loa22优势T细胞和B细胞（ T／B）联合抗原表位及其免疫原性。方法采用PCR检测我国流行的8群8型株问号钩体loa22基因，T-A克隆后测序。构建问号钩体黄疸出血群赖型赖株loa22基因原核表达系统。 Ni-NTA亲和层析法提纯表达的目的重组蛋白rLoa22并制备其兔抗血清及其IgG。采用生物信息学软件预测Loa22的T-B联合抗原表位。采用噬菌体展示联合Western blot法、ELISA分别检测重组噬菌体PⅢ蛋白展示的T-B联合表位肽和人工合成T-B联合表位肽的免疫原性。采用MTS法和ELISA分别检测T-B联合表位肽诱导T细胞活化及其分泌IL-2、IL-4和IFN-γ情况。结果所有受检的致病性钩体株均能检出loa22基因，其核苷酸和氨基酸序列相似性高达85．5％～99．8％和93．9％～99．5％。所构建的loa22基因原核表达系统能高效表达rLoa22。 Loa22-77、Loa22-90、Loa22-125和Loa22-157这4个T-B联合抗原表位中，仅有Loa22-90显示了很强的Western blot阳性条带。 Loa22-90能有效诱导CD4＋T细胞增殖及IL-2（Th1）和IL-4（Th2）水平显著升高（P＜0．05）。结论 Loa22是问号钩体序列保守的属特异性外膜蛋白抗原，其优势T-B联合抗原表位为Loa22-90，该表位可作为钩端螺旋体多抗原肽疫苗的候选表位。
목적：사선병감정치병성문호구단라선체（간칭구체）외막단백Loa22우세T세포화B세포（ T／B）연합항원표위급기면역원성。방법채용PCR검측아국류행적8군8형주문호구체loa22기인，T-A극륭후측서。구건문호구체황달출혈군뢰형뢰주loa22기인원핵표체계통。 Ni-NTA친화층석법제순표체적목적중조단백rLoa22병제비기토항혈청급기IgG。채용생물신식학연건예측Loa22적T-B연합항원표위。채용서균체전시연합Western blot법、ELISA분별검측중조서균체PⅢ단백전시적T-B연합표위태화인공합성T-B연합표위태적면역원성。채용MTS법화ELISA분별검측T-B연합표위태유도T세포활화급기분비IL-2、IL-4화IFN-γ정황。결과소유수검적치병성구체주균능검출loa22기인，기핵감산화안기산서렬상사성고체85．5％～99．8％화93．9％～99．5％。소구건적loa22기인원핵표체계통능고효표체rLoa22。 Loa22-77、Loa22-90、Loa22-125화Loa22-157저4개T-B연합항원표위중，부유Loa22-90현시료흔강적Western blot양성조대。 Loa22-90능유효유도CD4＋T세포증식급IL-2（Th1）화IL-4（Th2）수평현저승고（P＜0．05）。결론 Loa22시문호구체서렬보수적속특이성외막단백항원，기우세T-B연합항원표위위Loa22-90，해표위가작위구단라선체다항원태역묘적후선표위。
Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.