Biomedical subjects

Hong Gao

Publications and source records attributed to Hong Gao.

At least 73 records · Page 4Linked to original sources

PubMed2004

New mutation points in 23S rRNA gene associated with Helicobacter pylori resistance to clarithromycin in northeast China.

AIM: To investigate the resistance rate of Helicobacter pylori (H pylori ) to clarithromycin, metronidazole, amoxicillin and tetracycline to guide clinical practice, and to study the mechanism of H pylori resistant to clarithromycin. METHODS: Thirty H pylori strains were isolated from the mucosa of peptic ulcer, gastric tumor and chronic gastritis patients, then the minimal inhibitory concentration (MIC) to clarithromycin, metronidazole, amoxicillin and tetracycline was evaluated by E-test method. The sequence analysis of PCR fragments was conducted in 23S rRNA gene of H pylori resistant to clarithromycin to get the resistance mechanism of the bacteria. RESULTS: Among 30 H pylori strains, 7 cases were resistant to clarithromycin, 12 to metronidazole, 2 to tetracycline and no strain was found to be resistant to amoxicillin. The resistance rates were 23.3%, 40%, 6.7% and 0%, respectively. Three new mutation points were found to be related to the clarithromycin resistance in H pylori isolates, which were G2224A, C2245T and T2289C. CONCLUSION: In northeast China, H pylori shows high resistance to metronidazole, while sensitive to amoxicillin. The mechanism of resistance to clarithromycin may be related to the mutation of G2224A, C2245T and T2289C in the 23S rRNA gene.

Anti-Bacterial Agents↗

PubMed2004

Coadministration of a liver X receptor agonist and a peroxisome proliferator activator receptor-alpha agonist in Mice: effects of nuclear receptor interplay on high-density lipoprotein and triglyceride metabolism in vivo.

Liver X receptors (LXRs) are master transcription factors regulating cholesterol and fatty acid metabolism. Treatment of C57B6 mice with a specific synthetic LXR agonist, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317), resulted in elevated high-density lipoprotein (HDL) cholesterol as well as plasma and liver triglycerides. Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists are known to induce peroxisomal fatty acid beta-oxidation and also mediate HDL cholesterol metabolism. We have explored the hypothesis that simultaneous activation of PPARalpha and LXR may lead to additive effects on HDL cholesterol elevation as well as attenuation of triglyceride accumulation. Coadministration of T0901317 and the specific PPARalpha agonist [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy14643)] in mice led to synergistic elevation of HDL cholesterol that was primarily associated with enlarged HDL particles enriched with apoE and apoAI. Liver phospholipid transfer protein (PLTP) mRNA and plasma PLTP activity were additively elevated, suggesting a role of PLTP in the observed HDL cholesterol elevation. Moderate increases in plasma triglyceride levels induced by LXR activation was reduced, whereas the accumulation of triglyceride in the liver was not altered upon coadministration of the PPARalpha agonist. Peroxisomal fatty acid beta-oxidation in the liver was dramatically elevated upon PPARalpha activation as expected. Interestingly, activation of LXRs via T0901317 also led to a significant increase in peroxisomal fatty acid beta-oxidation. Sterol regulatory element binding protein 1c expression was dramatically up-regulated by the LXR agonist but was not changed with PPARalpha agonist treatment. Liver lipoprotein lipase expression was additively increased upon LXR agonist and PPARalpha agonist coadministration. Our studies mark the first exploration of nuclear receptor interplay on lipid homeostasis in vivo.

Animals↗

PubMed2004

Construction, expression and identification of a recombinant BCG vaccine encoding human Mycobacterium tuberculosis heat shock protein 65.

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↗

PubMed2004

An expression profile of human alpha-lactalbumin in the milk of transgenic mouse.

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5' flanking region and a 2.0-kb coding region of human alpha-lactalbumin, as well as a 227-bp 3'-flanking region from bovine growth hormone gene. A founder female expressed human alpha-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total alpha-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the halpha-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of alpha-lactalbumin might boost more milk output.

Animals↗

PubMed2004

Expression of lung resistance-related protein in transitional cell carcinoma of bladder.

OBJECTIVES: To determine the role of lung resistance-related protein (LRP) in intrinsic multidrug-resistance (MDR) of bladder cancer. METHODS: The study group consisted of 66 patients with newly diagnosed primary bladder cancer. No patient had been treated preoperatively with either radiotherapy or chemotherapy. Reverse transcriptase-polymerase chain reaction was performed to measure LRP, multidrug resistance-associated protein 1 (MDR1), and MRP1 mRNA expression. The expression of LRP, p53 proteins, and p63 proteins was examined by immunohistochemistry. We analyzed the correlation of LRP with the above indexes and the clinical pathologic parameters. RESULTS: The expression rate of LRP mRNA (63.6%) was the greatest among the three MDR markers in primary bladder cancer without exposure to chemotherapy. The LRP mRNA level was significantly greater in normal bladder tissue than in transitional cell carcinoma bladder tissue (P <0.01) and in superficial cancer than in invasive cancer (P = 0.013). LRP mRNA expression showed no association with either MDR1 or MRP1, but close correlation with the LRP level (P = 0.001). LRP was associated with low-grade (P <0.01) and low-stage (P <0.05) cancer but had no association with tumor suppressor p53 or p63. CONCLUSIONS: The grade and stage-related expression pattern of LRP indicates that it may be a predictive index for intrinsic MDR in early bladder cancer. Anticancer drugs out of the MDR spectrum of LRP may be more effective for patients with early bladder cancer.

Biomarkers, Tumor↗

PubMed2004

Analysis of unknown organic pollutants in sewage by solid-phase extraction combined with gas chromatography-mass spectrometry.

A rapid, simple, and reliable method of solid-phase extraction (SPE) combined with gas chromatography (GC)-mass spectrometry (MS) is developed for the analysis of a wide range of polarity of unknown organic pollutants in sewage. Wastewater samples are extracted by passing them through disposable C(18) cartridges, and the extracts are then analyzed by GC-MS. Different SPE parameters for ten organic compounds in the list of priority pollutants suggested by the China Environmental Protection Agency (EPA) are studied, and their breakthrough volumes are determined. Extraction recoveries for the tested compounds are greater than 60%, except the recovery of 1,2-dichloroethane is 48%. The relative standard deviations are less than 7.8% (n = 3). The developed approach is successfully applied for the identification of organic components in a sewage sample. Over 220 organic pollutants are identified, with 5 of these present in the list of priority pollutants suggested by the U.S. EPA and 4 from the list by the China EPA.

Environmental Pollutants↗

PubMed2004

Importance of the B ring and its substitution on the alpha-glucosidase inhibitory activity of baicalein, 5,6,7-trihydroxyflavone.

Hydroxychromones and B-ring-substituted 5,6,7-trihydroxyflavones were prepared to evaluate the contribution of the B ring of baicalein (5,6,7-trihydroxyflavone, 1) to its potent alpha-glucosidase inhibitory activity. Hydroxychromones, which lack 6-hydroxyl substitution, did not show any inhibitory activity, while 5,6,7-trihydroxy-2-methylchromone (5) showed high activity. Among the tested B-ring-substituted 5,6,7-trihydroxyflavones, the 4'-hydroxy-, 3',4'-dihydroxy-, and 3',4',5'-trihydroxy-substituted derivatives were found to give more activity than that of 1. The methoxy-substituted derivatives, however, showed less activity than 1. The results suggest that the B ring of 1 was not essential, although advantageous to the activity; hydroxyl substitution on the B ring of 5,6,7-trihydroxyflavones was favorable to the activity, whereas methoxyl substitution was unfavorable; at least 4'-hydroxyl substitution of 5,6,7-trihydroxyflavones was required for enhanced activity, in which the number of hydroxyl groups did not take part.

Animals↗

PubMed2004

Structure-activity relationships for alpha-glucosidase inhibition of baicalein, 5,6,7-trihydroxyflavone: the effect of A-ring substitution.

In order to estimate the effects of the A-ring hydroxyl group of baicalein (5,6,7-trihydroxyflavone, 1) on rat intestinal alpha-glucosidase inhibition, flavone, monohydroxyflavones, dihydroxyflavones, and methylated derivatives of 5,6,7-trihydroxyflavone were used for the structure-activity relationship (SAR) study. The importance of the 6-hydroxyl group of baicalein was validated for an exertion of the activity. And also, the tested flavones which lacked a hydroxyl substituent on any of positions 5, 6, or 7, showed no activity. Hence, the 5,6,7-trihydroxyflavone structure was concluded to be crucial for the potent inhibitory activity. In addition, an introduction of electron-withdrawing or electron-donating groups at position 8 of baicalein led to a dramatic decrease for activity, except for 8-fluoro-5,6,7-trihydroxyflavone, which carried a less bulky substituent on position 8. Hence, this result suggested that a sterically bulky substituent on C-8 of baicalein was detrimental for the activity regardless of its electronic nature. Through examining the inhibitory mechanism of baicalein against rat intestinal alpha-glucosidase, it was suggested to be a mixed type inhibition.

Animals↗

PubMed2004

Effect of human osteopontin on proliferation, transmigration and expression of MMP-2 and MMP-9 in osteosarcoma cells.

BACKGROUND: To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro. METHODS: The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E. coli BL21 (DE3) cells and purified with ProBond trade mark Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel. RESULTS: The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells. CONCLUSION: hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.

Bone Neoplasms↗

PubMed2004

[Detection of mutation in exon 5 and exon 8 of PTEN in laryngeal squamous cell carcinoma].

OBJECTIVE: To detect mutations of exon 5 and exon 8 of phosphatase and tensin homology deleted on chromosome10/mutated in multiple advanced cancer1/TGF-beta regulated and epithelial cell-enriched phosphatase (PTEN/MMAC1/TEP1 for short PTEN) gene in laryngeal squamous cell carcinoma (LSCC) and analyze the relationship between the mutation and LSCC. METHODS: Fresh tumor samples from 40 LSCC patients were examined using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequencing. RESULTS: The mutation of exon 5 and exon 8 occurred in supra-glottic laryngeal carcinoma and no mutation in the glottic laryngeal carcinoma was found. There were 6 cases of mutation for exon 5, and the mutation rate was 15%. In 2 cases, 1 base pair insertion TT-->TAT in codon 85, while in other two cases, 1 base pair deletion GCA-->GC in codon 86, and the two type mutation may result in frame shift mutation. One case had the above two type mutation simultaneously, which may result in missense mutation; 1 case had 1 base pair insertion TT-->TAT in 85 codon and 1 base pair deletion GCA-->GA, which may result in nonsense mutation. The pathology of 3/6 (50%) cases was low differentiation, and the others (3/6, 50%) were middle differentiation. Five cases had lymphatic metastasis, and the rate of which was 83.3%; the other one had no lymphatic metastasis, and the rate of which was 16.7%. There was only 1 case mutation for exon 8, and the mutation rate was 2.5%. One base pair deletion AAA-->AA in 276 codon as well as 1 base pair insertion CT-->CCT between the 336 codon and 337 codon may result in frame shift mutation. The pathology of the case was low differentiation. CONCLUSION: The codon 85,86 of PTEN gene in exon 5 may be "hot spots" in LSCC and the mutation of PTEN gene may be related with the lymphatic metastasis and middle or low differentiation.

Adult↗

PubMed2004

[Study of the effects of utero-placental ischemia on the body and nervous system development in fetal rats].

OBJECTIVE: To discuss the effects of utero-placental ischemia on the body and nervous system development in fetal rats. METHODS: By clamping the unilateral uterine artery of the rat, we produced a utero-placental ischemia model. The opposite uterus of the rat with normal uterine artery supply served as control. We compared the body weight, weight of brain, and the expression of growth associated protein-43 (GAP-43) mRNA in cerebral tissue by RT-PCR in the 13 day (group 1) and 17 day (group 2) old fetal rats respectively. RESULTS: The body weight and weight of brain in group 1 were 3.2 g and 0.16 g respectively, significantly lower than those of control 1 of 3.6 g and 0.18 g respectively (P < 0.01, P < 0.05). The body weight and weight of brain in group 2 were 3.1 g and 0.16 g respectively, also significantly lower than those of control 2 of 3.72 g and 0.17 g respectively (P < 0.01, P < 0.05). The expression of GAP-43 mRNA in cerebral tissue of the group 1 (1.10) and its control (1.12) showed no obvious difference (P > 0.05). However, GAP-43 mRNA in cerebral tissue of the group 2 (1.06) was significantly decreased compared with that of its control (1.21) (P < 0.01). CONCLUSION: Clamping the uterine artery can mimic intrauterine ischemia and produce FGR model. Intrauterine ischemia in the late stage of pregnancy may decrease the expression of GAP-43 mRNA, which might affect the development of nervous system in rats.

Animals↗

PubMed2004

Establishment of a cell-based assay to screen regulators for Klotho gene promoter.

AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS: The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment. The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2 effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

Aging↗

PubMed2004

[Altered expression of PTEN gene and LOH of its epigenetic microsatellite in gastric carcinoma].

OBJECTIVE: To investigate the expression of PTEN and loss of heterozygosity (LOH) of its epigenetic microsatellite in gastric carcinoma and explore their roles in progression of gastric carcinoma. METHODS: LOH of epigenetic microsatellites of PTEN (D10S541, D10S583 and D10S1687) in advanced gastric cancer was detected by PCR-SSCP. Expression of PTEN mRNA and protein in normal gastric mucosa and gastric cancer was evaluated by RT-PCR and SABC immunohistochemistry, respectively. The relationship between expression of PTEN mRNA and protein and lymph node metastasis or LOH of microsatellites was discussed. RESULTS: LOH of D10S541, D10S583 and D10S1687 was found in 37.5% (21/56) of advanced gastric cancers. The positive rates of PTEN mRNA expression were 80.4% (45/56), 45.5% (5/11) and 32.1% (18/56) in normal mucosa, early and advanced gastric carcinomas, respectively, while 78.6% (44/56), 44.5% (5/11) and 28.6% (16/56) at the protein level. PTEN mRNA and protein were less frequently expressed in early and advanced gastric carcinomas than that in normal gastric mucosa (P < 0.05). There was positive correlation between PTEN mRNA expression and LOH of microsatellites in advanced gastric carcinomas. PTEN protein expression paralleled with its mRNA expression (P < 0.05). The expression of PTEN mRNA and protein was negatively correlated with lymph node metastasis of advanced gastric carcinomas (P < 0.05). CONCLUSION: Down-regulated expression of PTEN gene is found in different stages of gastric carcinoma, and is closely correlated with LOH of its epigenetic microsatellites, which probably is its underlying molecular mechanisms. It suggests that altered PTEN gene contributes to tumorigenesis and progression of gastric carcinomas.

Gastric Mucosa↗

PubMed2004

[A comparative study on soil fauna in native secondary evergreen broad-leaved forest and Chinese fir plantation forests in subtropics].

In this study, we investigated the response of soil animal communities to the replacement of native secondary forest by Chinese fir plantation forest and successive rotation of Chinese fir in subtropics. Three adjacent forest stands, i.e., native secondary evergreen broad-leaved forest stand (control) and Chinese fir plantation stands of first (20 yr) and second (20 yr) rotations were selected for the comparison of soil fauna. All animals were extracted from the floor litter and 0-15 cm soil layer of the stands in Summer, 2003 by using Tullgren method, wet funnel method and hand-sorting method. Compared to two Chinese fir plantation forests, the native secondary evergreen broad-leaved forest had a higher abundance and a higher taxonomic diversity of animals in soil and litter, but there were no significant differences in the biomass and productivity of soil fauna between all study stands. The abundance or diversity did not differ significantly between the first rotation and second rotation stands, too. The results supported that vegetation cover might be one of the main forces driving the development of soil animal communities, and the effect of successive rotation of Chinese fir on the development of soil fauna was a slow-running process.

Animals↗

PubMed2004

[An overview on studies of soil organic matter in Chinese fir plantation].

Soil organic matter (SOM) is an important component of soil and an important carbon pool of terrestrial ecosystems, which plays an important role in soil nutrient cycling and in the balance of global carbon cycle. This paper discussed the SOM quality in Chinese fir plantation, the influence of rotation on SOM content, the change of SOM during Chinese fir growth, and the effect of human activities such as burning, site preparations and fertilization on SOM. Human activities changed soil environment including soil moisture, temperature, total porosity and nutrient contents, and hence, made the turnover of SOM slower or quicker. The content and quality of SOM in mixed forests were higher than those in pure Chinese fir plantations, and decreased with rotation. In the last part of this paper, some issues to be further researched were put forward, including the turnover model of SOM, its components, and its relationship with global carbon cycle.

Carbon↗

PubMed2004

[Clonality detection of cervical cancer and cervical intraepithelial neoplasia by human androgen receptor gene analysis].

OBJECTIVE: To assess the clonality of cervical cancer and cervical intraepithelial neoplasia (CIN) by the study of X-chromosome inactivation pattern with human androgen receptor gene (HUMARA) analysis. METHODS: Tissue DNA from samples of 26 invasive cervical cancers (9 in stage I, 10 in stage II and 7 in stage III), 31 CINs (12 CINI, 10 CIN II and 9 CIN III) and 33 normal cervixes was extracted, and then digested with methylation sensitive restriction endonuclease Hha I. HUMARA fragment was amplified with PCR method and PCR product was electrophoresed. Clonality was assessed by observing the electrophoresis bands. RESULTS: Heterozygotic rate of HUMARA was 89%. Monoclonal rates of cervical cancer, CIN, and normal cervix were 92%, 38% and 10%, respectively. Differences between cervical cancer and CIN and between cervical cancer and normal cervix were both extremely statistically significant (P < 0.001). Difference between CIN and normal cervix was also statistically significant (P = 0.012). Monoclonal rate of cervical cancer in stage I, II, III was 6/7, 90% and 7/7 respectively, without significant difference between them (P > 0.05). Monoclonal rate of CINI, II, III was 20%, 38% and 63% respectively, showing increasing tendency with grade, but without significant difference between them (P > 0.05). CONCLUSIONS: Invasive cervical cancer is a result of clonal proliferation of cancer cells. Active intervention or close follow up should be given to CIN with monoclonality.

Adult↗

PubMed2004

[Soil quality assessment of forest stand in different plantation esosystems].

After a clear-cutting of the first generation Cunninghamia lanceolata plantation in 1982, three plantation ecosystems, pure Michelia macclurei stand (PMS), pure Chinese-fir stand (PCS) and their mixed stand, were established in spring 1983, and their effects on soil characteristics were evaluated by measuring some soil physical, chemical, microbiological and biochemical parameters. After 20 years' plantation, all test indices showed differences among different forest management models. Both PMS and MCM had a favorable effect on soil fertility maintenance. Soil quality assessment showed that some soil functions, e.g., water availability, nutrient availability, root suitability and soil quality index were all in a moderate level under the mixed and pure PMS stands, whereas in a relatively lower level under successive PCS stand. The results also showed that there existed close correlations between soil total organic C (TOC), cation exchange capacity (CEC), microbial biomass-C (Cmic) and other soil physical, chemical and biological indices. Therefore, TOC, CEC and Cmic could be used as the indicators in assessing soil quality in this study area. In addition, there were also positive correlations between soil microbial biomass-C and TOC, soil microbial biomass-N and total N, and soil microbial biomass-P and total P in the present study.

Cunninghamia↗

PubMed2004

[Construction of a secreting prokaryotic shuttle expressing plasmid pBCG-sP-HSP65 and its expression in Mycobacterial smegmatis].

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↗