To construct the secretive prokaryotic shuttle expression plasmid pBCG-SP-HSP65, the signal peptide sequence of antigen 85B amplified from Bacillus Calmette-guérin (BCG) genome by PCR and the whole HSP65 DNA sequence of human M. tuberculosis obtained from the plasmid pCMV-MTHSP65 by PCR were cloned into the plasmid pBCG-2100 under the control of the promoter of Heat Shock Protein 70 (HSP70) from human M. tuberculosis. Recombinants were electroporated into Mycobacterial smegmatis and induced by heating. Results of the induced expression were detected by SDS-PAGE and the biological activity of the expressed protein was tested by Western-blot analysis. Results showed pBCG-SP-MTHSP65 was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and DNA sequencing analysis. After it was electroporated into Mycobacterial smegmatis and induced by heating, the percentage of expressed 65kD protein in Mycobacterial smegmatis detected by SDS-PAGE was 20% in total bacterial protein. But the percentage of expressed 65kD protein in recombibinant Mycobacterial smegmatis was up to 34.46% in total bacterial protein and 68.56% in the total protein of cell lysate supernants, Which demonstrated the recombinant HSP65 gene could express in recombinant with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive proteins could specially combine with antibody against human M. tuberculosis HSP65. Orally, pBCG-SP-HSP65 was successfully constructed; HSP65 gene could express in Mycobacterial smegmatis with high efficiency via it. And the expressed proteins possess the biological activity. So it provids experimental evidence for the application of the recombinant Mycobacterial smegmatis and the development of the vaccine against tuberculosis.
Bacterial Proteins ; biosynthesis ; genetics ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Mycobacterium smegmatis ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

BACKGROUND: Nontuberculous mycobacteria (NTM) should be correctly identified to the species level, because of different treatment plans among NTM species. This study was performed to assess the usefulness of real-time PCR and melting curve analysis in the identification of NTM. METHODS: One hundred fifty-two clinical NTM isolates were identified to the species level by PCR-restriction fragment length polymorphism analysis (PRA). Those strains were then identified by multiplex real-time PCR and melting curve analysis on the 16S rRNA gene and hsp65 gene. RESULTS: In the 16S rRNA gene fragment analysis, M. abscessus-M. chelonae group showed melting point at temperatures above 65 degrees C and M. avium complex (MAC; M. avium and M. intracelluare) below 48 degrees C, which differentiated M. abscessus-M. chelonae group and MAC from other NTM. In the hsp65 gene fragment analysis, M. abscessus-M. chelonae group was clearly divided into M. abscessus type I, M. abscessus type II, and M. chelonae according to the melting points at 61.25 degrees C, 66.06 degrees C, and 57.58 degrees C, respectively. CONCLUSIONS: With the multiplex real-time PCR and melting curve analysis of 16S rRNA and hsp65 genes, M. abscessus and M. chelonae were readily identified and MAC were differentiated from other NTM. Especially, M. abscessus and M. chelonae, which were not differentiated from each other with the 16S rRNA gene fragment analysis, were identified with hsp65 gene fragment analysis.
Bacterial Proteins/genetics ; Chaperonins/genetics ; Computer Systems ; DNA, Bacterial/chemistry ; Mycobacteria, Atypical/genetics/*isolation & purification ; Nucleic Acid Denaturation ; Polymerase Chain Reaction/*methods ; RNA, Ribosomal, 16S/genetics

OBJECTIVETo study the influence of hepatitis B virus (HBV) replication and expressions of different viral genes on CDC37 level in hepatocytes.

METHODSWe amplified and cloned 6 HBV genes (P, preS1, preS2, S, C and X) into pCMV expression vectors, which were transfected in Huh7 and HepG2 hepatoma cell lines, and CDC37 expression level in the cells was detected using Western blotting. Wealso cloned the promoter sequence of CDC37 into pGL3 vector, and co-transfected pGL3 with pCMV recombinant plasmids into Huh7 and HepG2 cells and the fluorescent signals were detected. To study the influence of HBV replication on CDC37 expression, we constructed 1.28-copy overlength genomes of HBV genotypes B, C, D and CD recombinant. The overlength HBV genomes were transformed into Adeasier-1 cells for recombination and into 293 cells for packaging. Huh7 and HepG2 cell lines infected with the packaged HBV recombinant adenoviruses were examined for CDC37 expression with Western blotting.

RESULTSWestern blotting showed that the expression of different HBV genes did not obviously affect the protein level of CDC37 in the hepatocytes. The protein expression of HBV genes had no effect on the activity of CDC37 promoter. Huh7 and HepG2 cells infected with 1.28-copy HBV replicon showed no significant changes in the expression level of CDC37.

CONCLUSIONHBV replication and its gene expression have no effect on the level of CDC37 in hepatocytes in vitro.

Adenoviridae ; Cell Cycle Proteins ; metabolism ; Chaperonins ; metabolism ; Gene Expression Regulation, Viral ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; virology ; Humans ; Transfection ; Virus Replication

BACKGROUNDNontuberculous mycobacterium (NTM) had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum (M. marinum) are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections.

METHODSSpecimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction (PCR) was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein (hsp) 65 kD gene.

RESULTSThe 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis (PRA) identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment.

CONCLUSIONSPRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.

Adolescent ; Adult ; Bacterial Proteins ; genetics ; Chaperonin 60 ; Chaperonins ; genetics ; Female ; Granuloma ; microbiology ; Humans ; Male ; Mycobacterium marinum ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Skin Diseases, Bacterial ; diagnosis ; Staining and Labeling ; Swimming Pools

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.
BCG Vaccine ; biosynthesis ; immunology ; Bacterial Proteins ; biosynthesis ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; immunology ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Mycobacterium tuberculosis ; genetics ; immunology ; Plasmids ; genetics ; Sequence Analysis, DNA ; Vaccines, Synthetic ; biosynthesis ; immunology

To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mycobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.
Animals ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; immunology ; Diabetes Mellitus, Type 1 ; prevention & control ; Escherichia coli ; genetics ; metabolism ; Female ; Heat-Shock Proteins ; genetics ; immunology ; Immunization ; Mice ; Mice, Inbred NOD ; Mycobacterium bovis ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

OBJECTIVETo screen the genes related to the occurrence and development of varicosis of the great saphenous vein in the patients with primary deep vein valve insufficiency.

METHODSUsing mRNA fluorescent differential display reverse transcriptive polymerase chain reaction (FDD-RTPCR), different genes expressed in the varicose great saphenous veins in patients with primary deep vein valve insufficiency and corresponding normal human tissues were compared. Differentially expressed cDNA fragments confirmed by Northern blot were compared and then cloned into the pGEM-Teasy vector. Positive clones were selected and sequenced. All the sequences were put into GenBank and analyzed by BLASTN software to search for their genetic origins.

RESULTSAltogether 37 different cDNA fragments were obtained and 30 of which were confirmed by Northern blot. Analysis of the sequences by BLASTN software showed that C(610) fragment (NO. 18 cDNA clone) shared 96% homology with the mRNA sequence of the human Mckusick-Kaufman syndrome gene (MKKS gene).

CONCLUSIONC(610) fragment is highly homologous with the mRNA sequence of the human MKKS gene and is closely related to the development of varicosis of the great saphenous vein in patients with primary deep vein valve insufficiency.

Base Sequence ; Blotting, Northern ; Cloning, Molecular ; Group II Chaperonins ; Humans ; Lower Extremity ; blood supply ; Molecular Chaperones ; genetics ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Saphenous Vein ; Sequence Homology, Nucleic Acid ; Varicose Veins ; etiology ; genetics ; Venous Insufficiency ; complications ; genetics