在日本鳗鲡(Anguilla japonica)中克隆获得2个γ-干扰素诱导的溶酶体巯基还原酶(GILT)基因AjGILT-1和AjGILT-2,并对其序列进行生物信息学分析,进而利用实时荧光定量PCR分别检测2个基因在不同组织器官中的表达水平,及其在脂多糖(LPS)、聚肌胞苷酸(PolyI:C)刺激和迟缓爱德华氏菌(Edwardsiella tarda)感染后的表达变化.结果显示:2个基因编码的蛋白序列中均含有GILT家族的特征序列CQHGX2 ECX2 NX4C、活性位点CXXC、2个糖基化位点和11个保守的半胱氨酸残基,基因组序列均含7个外显子和6个内含子.2个基因启动子中都存在多个转录因子结合位点,如)-干扰素活化位点以及特化蛋白l结合位点;不同的是AjGILTl启动子区存在多个核因子-1、激活蛋白-1结合位点及于扰素刺激反应元件,而在AjGILT-2启动子区未发现.AjGILT-1在鳃和肠中表达量最高,而AjGILT-2在性腺、头肾和脾脏中表达量最高;除肠道外,同一组织中AjGIT-2的表达量均高于AjGILT-1.LPS、PolyI:C刺激和E.ta rda感染后,AjGILT-1与AjGILT-2的表达在鳃中仅有少数变化,而在头肾和中肾中则变化较明显.综上所述,AjGILT-1和AjGILT-2基因可能参与了日本鳗鲡受病毒和细菌感染后的免疫应答.

Objective To clone and express the extracelluar region of TGF-β receptor-I in Schistosoma japonicum(SjTβRIout),and to identify its immunologic functions.Methods Part of the coding region of the SjTβRI gene was obtained by rapid amplification of cDNA ends(RACE).The extracelluar fragment of SjTβRI gene was amplified by RT-PCR,cloned into the prokaryotic expression vector pET28a(+),and then confirmed by sequencing method.The plasmid was then transfected into E.coli and the expression was induced by IPTG.The recombinant protein was purified using Ni-ion affinity chromatography.SD rats were immunized with the purified protein and Freund's adjuvant.ELISA was used to detect the production of the SjTβRIout specific antibody.Results The pET28a(+)-SjTβRIout recombinant expression plasmid was successfully constructed.Sequence analysis showed that the SjTβRIout gene had 399 bp(encoding 133 amino acids) and the gene was highly homologous with Schistosoma mansoni TGF-β receptor I.SjTβRIout recombinant protein with a molecular weight of 20 kDa was expressed in E.coli.The recombinant protein immunized SD rats produced a high titer( 1:100 000 000) of antigen-specific IgG antibody.Conclusion The pET28a(+)-SjTβRIout recombinant protein was proved to be immunogenic.

To study the SjTβRII-like gene from Schistosoma japonicum (S. japonicum), the coding region of SjTβRII-like was obtained by RACE and the extracellular fragment of SjTβRII-like gene was cloned into the prokaryotic expression vector pET28a (+) then expressed in E.coli by IPTG induction. The recombinant protein was detected by SDS-PAGE. SD rats were immunized with the purified recombinant protein in order to obtain the antisera and analyze its immunogenicity and antigenicity by Western blotting.It was found that the encoding sequence of the novel SjTβRII-like gene was 2283 bp (encoding 760 amino acids). Recombinant expression plasmid was successfully constructed, which could elicit specific antibodies efficiently in SD rats, indicating its immunogenicity and antigenicity. Results suggested that the extracelluar fragment of the novel SjTβRII-like gene of S. japonicum could be expressed in the prokaryotic system with immunological and antigenic activities efficiently.

Objective To analyze the PPIase activity of purified GST-SjFKBP12 recombinant fusion protein and to compare the transcription level of SjFKBP12 with Sj26GST.Method FKBP12 cDNA obtained from adult worm was cloned into pGEX-4T-1 vector and expressed.PPIase activity of the purified recombinant fusion protein was analyzed.Semi-quantitative RT-PCR was used to analyze the transcription level of SjFKBP12 and Sj26GST gene.Result SjFKBP12 cDNA was successfully cloned into pGEX-4T-1 vector.The purified GST-SjFKBP12 recombinant fusion protein was found to exhibit PPIase activity.Transcription level of SjFKBP12 in cercariae and eggs is 1.5 times higher than adults.Conclusion Functional GST-SjFKBP12 recombinant fusion protein was produced.High level of transcription of FKBP12 was detected in cercaria and eggs.The results may lay the foundation for the future vaccine development.