OBJECTIVE To study the molecular mechanism of the drug-resistance of Gram-negative bacilli to the third generation of cephalosporins.METHODS MICs of 13 ?-lactams to the eleven Gram-negative bacilli clinical isolates were detected with the standard agar dilution technique.K-B disc confirmatory method was conducted to determine the ESBLs phenotype of the eleven isolates.The ESBLs encoding genes were analyzed by using PCR.RESULTS The eleven isolates were all resistant to the third generation of cephalosporins(MIC≥64 ?g/ml).Disk confirmatory test showed that 10 isolates produced ESBLs.The hydrolytic activity of the ESBLs from the 10 isolates to cefoperazone and cefamandole was very high.However,the hydrolytic activity of the ESBLs from the 10 isolates to ceftazidime was very low.CONCLUSIONS The enzyme activities and the genes of extended-spectrum ?-lactamases from 10 Gram-negative bacilli clinical isolates are preliminarily analyzed.These results provide the basis for further study on the molecular mechanism of the drug-resistence of Gram-negative bacilli.

OBJECTIVE:To clone,prokaryotic express and characterize the TEM-type ?-lactamase produced by Enterobacter cloacae clinical isolate EC002. METHODS: The drug susceptibility of Enterobacter cloacae clinical isolate EC002 was detected by agar double dilution,double disk screening and confirmatory test were employed to detect the ESBLs. The isoelectric point (pI) of enzyme was detected by isoelectric focusing electrophoresis (IEF),the genes were coded by PCR amplification enzyme,and the prokaryotic expression and phenotype of the TEM-type ?-lactamase were detected. RESULTS: Enterobacter cloacae EC002 were resistant to most of the ?-lactamases. Positive results were noted for the phenotype identification and plasmid conjugation test. IEF showed that Enterobacter cloacae EC002 produced two ?-lactamases with pI value at 8.7 and 5.4 respectively,which were confirmed to be CTX-M-22 and a new TEM-subtype ?-lactamase by DNA sequencing,and the phenotype of the expressed enzyme of the cloned strains was non-ESBLs. The TEM-type ?-lactamase was named as TEM-141 by GenBank. CONCLUSION: The TEM-141 produced by Enterobacter cloacae EC002 was a new type of plasmid-mediated broad-spectrum ?-lactamase.

Objective To study the molecular genetic mechanism of a patient with 17? hydroxylase (CYP17) deficiency. Methods Genomic DNA were abstracted from the blood of the patient, her parents and healthy control. The 8 exons of CYP17 gene were amplified, using 5 pairs of designed primers, with polymerase chain reaction (PCR), and the 8 exons were sequenced by the dideoxy terminator method to determined the mutation sites. The corresponding exons of the parents of the patients were also amplified and sequenced to determine the zygosity of the patient and the source of the gene variances. Results The analysis revealed that the patient (46, XY) was a compound heterozygote carrying two different inherited mutations on CYP17 gene, one from mother containing a point mutation Arg 96 (C G G)→ Gln(C A G) and the other from father containing a nine base deletion (CACTCTTTC) at amino acid position 487～489 (Asp Ser Phe) near the carboxyl terminus of P450c17. Conclusion The CYP17 gene of the patient with 17? hydroxylase deficiency is a compound heterozygous mutation. The mutation changes the amino acid sequence of P450c17 enzyme, which in turn affected the enzymatic activity. Arg 96 is essential in P450c17 enzyme activity. Deletion of Asp 487 Ser 488 Phe 489 in exon 8 may be a prevalent mutation causing P450c17 deficiency in Southeast Asia.

Objective To explore new method for enhancing the efficacy of tuberculosis DNA vaccine. Methods Two recombinant plasmids were constructed, one named as pBK GM/85A encoding mouse granulocyte macrophage colony stimulating factor(GM CSF) fused to Mycobacterium tuberculosis Ag85A antigen, the other named as pBK 85A encoding Mycobacterium tuberculosis Ag85A antigen alone. Subsequently, the two plasmids were transferred into cultured COS7 cells by using cationic liposomes. The expression products were identified by Western blotting. Then, in a murine model, we compared the immunogenicity and protective immunity of the two recombinant plasmids following genetic immunization. Results All pBK GM/85A injected mice elicited higher antibody titres than that for pBK 85A injected mice. Lymphocytes obtained from the spleen of pBK GM/85A immunized mice exhibited higher lymphocyte proliferative response、IFN ? production and CTL activity than that for pBK 85A immunized mice. The protective efficacy was also higher for pBK GM/85A immunized mice than that for pBK 85A immunized mice. However, The protective efficacy for pBK GM/85A immunized mice was lower than that for BCG immunized mice. Conclusion These results showed that DNA vaccines with GM CSF/antigen fusion constructs could greatly improve the immunogenicity of DNA vaccine against Mycobacterium tuberculosis.

AIM: To study the inhibitory effects of 10-23 deoxyribozyme (10-23 DRz) on Escherichia coli ?-lactamase gene expression. METHODS: According to the gene sequence of Escherichia coli ?-lactamase gene blashv-5, 10-23 DRz and antisense oligonucleotides (As-ODN) were designed and synthesized. 10-23 DRz, As-ODN or control oligonucleotides were respectively introduced into Escherichia coli by the method of electroporation. Following electroporation, bacterial viability in liquid medium contained ceftazidime was detected, bacterial ?-lactamase expression was analysed by using IEF-PAGE and the ?-lactamase band was measured with gel documentation-analyzing system. RESULTS: A_ 600 in 10-23 DRz transfected Escherichia coli was lower compared with that in As-ODN transfected Escherichia coli (P

AIM: Construction of an eukaryote- E. coli shuttle expressing recombinant plasmid which expresses OmpL1 envelope protein of pathogenic Leptospira, serovar Lai strain 017. METHODS: The OmpL 1 gene was amplified by PCR from the leptospiral genome. Then it was cut with restriction enzymes and ligated to the plasmid pBK-CMV. The correct recombinant plasmid was screened out with analysis of restriction enzymes and PCR. After inducing the E. coli baring recombinant plasmid with IPTG,the complete protein of the bacteria was extracted for SDS-PAGE. At the same time, OD600 of the host bacteria was examined at different time after inducing or uninducing with IPTG. RESULTS: Five strains E. coli containing proper recombinant plasmids were screened out. Four strains E. coli expressed a new protein with a weight of 37 kD among them. With the expression of the heterogenous protein,the OD600 of the host bacteria decreased. CONCLUSION: The shuttle expressing plasmid of the OmpL 1 gene of strong virulent Leptospira strain 017 was successfully constructed. Furthermore,the recombinant plasmid expressed the expected OmpL 1 fusion protein in E. coli and the expression of the heterogenous protein had toxic effect on the host bacteria. This work was important for the future research of OmpL1 protein which relates to the diagnosis,new vaccine preparing and the pathogenic mechanism of leptospirosis.

AIM:To detect the drug-resistance mycobacterium tuberculosis by using DNA microarray hybridization.METHODS: DNA microarray for detecting drug-resistance of mycobacterium tuberculosis was prepared; Clinical isolated strains were cultivated and their drug-resistance sensitivity was detected. The genome DNA of mycobacterium tuberculosis was prepared and the drug-resistance genes of the mycobacterium tuberculosis were amplified by PCR. Then the gene chip was hybridized, washed, detected and analyzed. RESULTS: Results of cultivating mycobacterium tuberculosis and detecting the drug-resistance sensitivity: one strain was drug-sensitive; four strains were multi-drug-resistant; The detecting results of the drug-resistance was consistent with the results of diagnosis therapy of the 5 clinical patients. The detecting results of gene chip confirmed the above facts. CONCLUSION: Detecting drug-resistance mycobacterium tuberculosis by the gene chip is precise, fast and highly-efficient.

OBJECTIVETo construct recombinant BCG against leptospirosis.

METHODSWe amplified the entire open reading frame of the OmpL1 gene from the genome of the leptospire serovar Lai strain 017. Two recombinant plasmids pBQ1 and pBQ2 were constructed by oriented ligation based on the E. coli-BCG shuttle plasmids pMV261 and pMV361 respectively. The recombinant plasmids were transformed into BCG by electroporation. The rBCGs bearing pBQ1 and pBQ2 were induced by high temperature of 45 degrees C.

RESULTSThe expressed product, a 35kD protein was detected by SDS-PAGE. The result indicates that pBQ1 and pBQ2 can express OmpL1 in rBCG.

CONCLUSIONThe technical methods in this study may help detect the immunogenicity and immunoprotection of OmpL1 and develop more safe, highly effective rBCG bearing leptospiral antigen with long-lasting protection.

BCG Vaccine ; genetics ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; DNA, Bacterial ; genetics ; Gene Expression ; Genes, Bacterial ; Leptospira interrogans ; genetics ; Open Reading Frames ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

【Objective】 To further determine the relationship between blood donor role cognition and blood donation behavior, then prepare of blood donor role identity scale(BDRIS). 【Methods】 The preparation of the scale was divided into five stages. It includes literature retrieval, expert interview, construction of the basic framework of the scale, scale test and statistical index test. The study focused on items selection, dimensions identification, measurement reliability, content and structural validity. 【Results】 The blood donor role identity scale consisted of 35 items. Exploratory factor analysis divided the items into six common factors, including role identity, role expectation, current situation of blood collection and supply, role conflict, external reward and blood donation experience. The α of scale was 0.840. 【Conclusion】 The reliability and validity of the scale meet the basic measurement requirements, and the framework of the blood donor role scale is basically formed. It can be used as one of the means to explore the ways and mechanisms of the role identity of blood donors in blood donation behavior.

Epidermal growth factor receptor (EGFR) mutations are the most common driver genes in the development of non-small cell lung cancer (NSCLC), of which mutations in exons 18-21 are frequent, especially the loss of exon 19 and exon 21 L858R mutation are the most frequent. Other rare gene mutations are rare. Simultaneous occurrence of two or more rare EGFR mutations are extremely rare in lung cancer, and the incidence of EGFR L833V/H835L rare gene compound mutations is very low, and there is little clinical data and evidence of relevant treatment methods. Some EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are effective in treating lung cancer patients with rare gene mutations. In this article, we reported a case of NSCLC patient with a rare gene compound mutation EGFR L833V/H835L, who responded to Afatinib in combination with Anilotinib treatment well after 5 months of treatment, and computed tomography (CT) showed shrinkage of lung lesions. Meanwhile, we also compiled previously reported NSCLC patients with EGFR L833V/H835L rare gene compound mutation and summarized the characteristics of this group of patients and the effect of applying different kinds of EGFR-TKIs treatment.
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Humans ; Adenocarcinoma of Lung/genetics* ; Carcinoma, Non-Small-Cell Lung/pathology* ; ErbB Receptors/genetics* ; Lung Neoplasms/pathology* ; Mutation ; Protein Kinase Inhibitors/therapeutic use*