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Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A

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World Journal of Gastroenterology 2004年第18期10卷 2747-2749页

作者： Si-WuFu Ya-LiZhang Dian-YuanZhou plainstitutefordigestivemedicine NanfangHospitalFirstMilitaryMedicalUniversityGuangzhou510515GuangdongprovinceChina

AIM: To establish an ELISA kit using monoclonal antibodies against Clostridium diffidle ( C. difficile) toxin ***: An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum,the wells were blocked with 100 g/L BSA in PBS-T. C. difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate ***: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli 2 strains of S. dysenteriae,1 strain of Bif. infantis, 5 strains of V. cholera, 2 strains of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/***: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

关键词： ELISA 单克隆抗体梭菌毒素A 血清

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pyloriconservative region of adhesin antigen and its immunogenicity

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World Journal of Gastroenterology 2004年第17期10卷 2498-2502页

作者： YangBai Ya-LiZhang Ji-DeWang Zhao-ShanZhang Dian-YuanZhou plainstitutefordigestivemedicine NanfangHospitaltheFirstMilitaryMedicalUniversityGuangzhou510515GuangdongProvinceChina InstituteofBiotechnology AcademyofMilitaryMedicalSciencesBeijing100071China

AIM: To construct a non-resistant and attenuated Salmonella typhimurium ( S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori(Hpylon) and evaluate its ***: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya1 delta Crp1 delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supematant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal ***: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully ***, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supematant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, bhe entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0×l0^10 cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune ***: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro,which providing a new live oral vaccine candidate for protection and care of H pylori infection.

关键词：沙门氏菌属盲肠疾病疲劳反应哈比特属幽门菌附着因子保守治疗抗原免疫原性消化系统

Expression of Helicobacter pyloriAlpA protein and its immunogenicity

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World Journal of Gastroenterology 2005年第15期11卷 2260-2263页

作者： JingXue YangBai YeChen Ji-DeWang Zhao-ShanZhang Ya-LiZhang Dian-YuanZhou plainstitutefordigestivemedicine NanfangHospitalTheFirstMilitaryMedicalUniversityGuangzhou510515GuangdongProvinceChina InstituteofBiotechnology AcademyofMilitaryMedicalSciencesBeijing100071China

AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent *** BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombina

关键词： H pylori Immunogenicity

Treatment of severe Clonorchiasis sinensis by endoscopic nasobiliary drainage and oral praziquantel

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World Journal of Gastroenterology 2004年第14期10卷 2150-2152页

作者： Fa-ChaoZhi Xu-MingLiu Ze-QuanLiu YanLin Shu-JianChen plainstitutefordigestivemedicine NanfangHospitalFirstMilitaryMedicalUniversityGuangzhou510515Guanl~donl~ProvinceChina DepartmentofDigestiveDiseases HexianHospitalPanyuGuangzhou511400GuangdongProvinceChina

AIM: To assess the therapeutic value of endoscopic nasobiliary drainage (ENBD) and oral praziquantel for severe Clonorchiasis sinensis ***: Of the 84 Clonorchiasis sinensis-infected patients enrolled, 58 were treated with ENBD (as observing group, ENBD group), 26 received operations (control group,operation group). Both of the two groups were comparable in terms of patient's age, body mass index. Before and one week after treatment, the average diameters of common bile ducts were measured by ultrasound, andserum bilirubin, ALP,γ-GT and ALT were detected by biochemical methods, After ENBD or operation, the patients took praziquantel for two days. RESULTS: Compared with the patients in operation group,ENBD patients in ENBD group had higher recovery rates of abdominal pain and fever as well as jaundice, quicker remission,smaller trauma, fewer complications and lower ***: ENBD combined with oral praziquantel is an effective and safe method for the treatment of severe Clonorchiasis sinensis.

关键词：治疗华支睾吸虫病内窥镜排脓法吡喹酮口服药

Cloning and expression and immunogenicity of Helicobacter pylori BabA_2 gene

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World Journal of Gastroenterology 2004年第17期10卷 2560-2562页

作者： YangBai Ya-LiZhang YeChen Jian-FengJin Zhao-ShanZhang Dian-YuanZhou plainstitutefordigestivemedicine NanfangHospitaltheFirstMilitaryMedicalUniversityGuangzhou510515GuangdongProvinceChina ChemistryuniversityofBeijing Beijing100071China InstituteofBiotechnology AcademyofMilitaryMedicalSciencesBeijing100071China

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori(H pylori) and to study the immunogenicity of ***: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) *** strain. Furthermore,BabA immunogenicity was studied by animal ***: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by *** BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize ***: BabA recombinant protein may be an potential vaccine for control and treatment of Hpyloriinfection.

关键词：无性繁殖基因表达免疫原性哈比特属幽门菌 BabA2基因重组细胞蛋白

Expression of Helicobacter pylori Hsp60 protein and its immunogenicity

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World Journal of Gastroenterology 2003年第12期9卷 2711-2714页

作者： YangBai Liang-RenLi Ji-DeWang YeChen Jian-FengJin Zhao-ShanZhang, Dian-YuanZhou Ya-LiZhang plainstitutefordigestivemedicine NanfangHospitalFirstMilitaryMedicalUniversityGuangzhou510515GuangdongProvinceChina ChemistryUniversityofBeijing Beijing100071China InstituteofBiotechnology AcademyofMilitaryMedicalScienceBeijing100071China

AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its ***: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal ***: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 ***: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

关键词：热休克蛋白60 幽门螺杆菌免疫原性 PCR DNA序列分析