Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator bacteria.

Female ; Genetic Predisposition to Disease ; Hepatitis B ; genetics ; Hepatitis B virus ; Humans ; Polymorphism, Genetic ; Pregnancy ; Pregnancy Complications, Infectious ; virology ; Promoter Regions, Genetic ; genetics ; Tumor Necrosis Factor-alpha ; genetics

Objective To compare diagnostic performance of ultrasound and radionuclide imaging in diagnosis of Meckel's diverticulum.Methods Totally 46 children suspected with Meckel's diverticulum were enrolled.Ultrasound,radionuclide imaging data were analyzed and compared with pathology.Results In 46 children suspected with Meckel's diverticulum,38 cases were confirmed by operation.Thirty-three cases of 38 were diagnosed Meckel's diverticulum by ultrasound,5 cases of 38 were false negative,there was no false positive case.Radionuclide imaging was positive in 24 cases of 46,false positive in 4 eases and false negative in 18 cases.The ultrasound diagnostic accuracy rate was 89.13％ (41/46),sensitivity was 86.84％ (33/38),specificity was 100％ (8/8).Diagnostic accuracy rate of radionuclide imaging was 52.17％ (24/46),sensitivity was 52.63 ％ (20/38) and specificity was 50.00 ％ (4/8).The sensitivity of ultrasound and radionuclide imaging in diagnosis of Meckel's diverticulum had significant difference (P＜0.01).Conclusion Ultrasound in diagnosis of Meckel's diverticulum has advantages of non-invasive,no radiation,acceptable price and high sensitivity.

Objective To investigate the efficacy and the mechanism of different dose hepatitis B immunoglohulin(HBIG)on prevention of HBV intrauterine infection and HBV S gene mutation. Methods HBV carrier mothers were randomly divided into three groups.Eighty-one HBsAg carrier pregnant women were divided into HBIG A group.HBIG B group and control group.Each subject in the HBIG A group received 200 U or 400 U(for HBsAg and HBeAg double positive carrier)intra muscularly at 3,2,1 month before delivery.Each subject in the HBIG B group received 200 U intra muscularly at 3,2,1 month before delivery.The subjects in the control group did not receive any treatment.Maternal blood samples were taken before HBIG injection and at delivery.Neonatal blood samples of all newborn infants after birth were taken before immunopropbylaxis.Their sera were ob tained to test HBV markers by enzyme immunoassay(EIA)and HBV DNA by fluorescence quantita- tive polymerase chain reaction(FQ-PCR),then to amplify and sequence HBV S gene region.Results The rate of HBV intrauterine infection in the HBIG group(14.5%)was lower than that in the control group(35.7%)(X~2=4.896,P=0.027).The rate of HBV intrauterine infection of newborns from HBsAg and HBeAg double positive carrier mother in the HBIG A group(37.5%)were lower than control group(100.0%)(X~2=7.273,P=0.007),while the rate was no different in the HBIG B group(71.5%)and the control group(X~2=2.637,P=0.104).Maternal HBsAg titer and HBV DNA level were of no difference among three groups before HBIG injection.Maternal HBsAg titers and HBV DNA levels of the HBIG A group were lower than those of the HBIG B group and the con- trol group at delivery.Among the 26 neonatal serum samples in the HBIG A group,10(38.5%)were positive for anti-HBs,while in the HBIG B group and in the control group,no neonatal serum sam- ples was positive.There was no significant difference of nucleotide and amino acid changes in the S gene between the HBIG group and the control group.Conclusions HBV infection in the uterus may be interrupted by injection HBIG intramuscularly before delivery.More efficacy would be found using variable HBIG dose according to different HBV virema and must be once more again injected just he- fore one week of delivery;anti-HBs transported to the fetus via the placenta and it's may be the im- portant mechanism of HBIG prevention.Asymptomatic HBsAg carrier mother received injections of HBIG before delivery should not influence HBV S gene mutation.Gene mutation of HBV is not the main factor in intrauterine transmission of HBV.

Objective To investigate the early-pathologic changes in children′s antrum infected with different types of Hp and study the Hp isolate′s pathogenic.Methods The serum types of CagA and VacA from Hp were determined by Westen-Blot in 70 patients with Hp positive and 36 patients with Hp negative.The standard of gastritis pathologic classification was accordance with that of international made in Sydney. The pathogenic of Hp affecting was evaluated by the degree of inflammation, severity of active gastritis,lymph follicles and atrophy.Results The detection rate of type Ⅰwith high virulence in Hp isolates was 68.1%,mid-type isolates was 27.7% and type Ⅱ with low virulence isolates was 4.2%.To observe the pathologic distinction in 49 patients with type Ⅰisolate,20 patients with mid-type isolate and 3 patients with type Ⅱ isolate,the type Ⅰ and mid-type isolates had significant difference in inflammation and their activity in either antrum or duodenal ampulla.Three patients with type Ⅱ isolate have not active gastritis.Type Ⅰand mid-type isolates had significant difference in lymph follicles,and the lymph follicles caused by type Ⅰwere significant higher than those caused by mid-type.But there were no significant differences in intestinal metaplasia and atrophy.Conclusions TypeⅠisolate with high virulence is the main detection isolate of children infected by Hp in our district.There is inflammation occurrence in antrum specimens in childhood who infected with Hp.

Objective To evaluate the using of either 225 or 150 microgrammes of mifepristone combined with misoprostol for termination of second-trimester gestation(16～24 weeks).Methods 180 women requesting voluntary induced abortion during gestation 16～24 weeks were randomised to three groups,group 1:oral mifepris- tone 225rag,group 2:oral mifepristone 150mg,and group 3:injected 100rag rivanot by amniocentestis.The total suc- cess rate,once success rate,the interval of having-medicine to uterine-constraction,the volume of bleeding within 2 hours after labour and cervical laceration rate were observed.Results The once success rate of induced labour in group 1 was higher than that in group 2 and group 3(P

Objective To study the application of multi-slice spiral CT(MSCT) portography in diagnonsis of pancreatic portal hypertension. Methods Forty-seven patients with lesion in body or tail of pancrease,47 normal subjects and 126 patients with portal hypertension underwent MSCT portography with LightSpeed 16 CT scanner. The inner diameter of portal system and the main collateral veins were measured in maximun intensity projection (MIP) image. The volume of liver and spleen were also measured in volume rendering (VR) image. The liver parenchyma and main portal vein enhancement in portal vein phase were also taken. The endoscopy examination was made in 57 patiens with portal hypertension. Results In 47 patiens with lesion in body or tail of pancreas , stenosis or occlusion of spleen vein were found in 38 patients(pancreatic portal hypertension in 27 patients, chronic and acute pancreatitis in 11 patients). In 38 patients with pancreatic portal hypertension, esophageal varices was found in 5 patients(13.2%), gastric fundus varix in 25 patients (65.8%), gastric body variees in 22 patients (57.9%), short-gastric vein/post-gastric vein(SGV/PGV) in 26 patients (68.4%), coronal gastric vein in 26 patients (68.4%),dilated gasto-omenta vein in 24 patients(63.2%), mesenterica varicesin 1 patient, splenic vein occlusion in 14 patients (36.8%), splenic vein stenosis in 23 patinets(63.2%). Conclusions The patients with pancreatic portal hypertension were demonstrate characteristic changes in MSCT portography. The MSCT portography is helpful in etiological diagnosis of pancreatic portal hypertension by supplying images in vessel morphology.

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways ; Ginsenosides ; biosynthesis ; Glycosyltransferases ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Synthetic Biology

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.
Fermentation ; Gene Dosage ; Glycosylation ; Molecular Chaperones ; Pichia ; metabolism ; Promoter Regions, Genetic ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

Antigens, CD34 ; metabolism ; Cytokines ; metabolism ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mucin-1 ; metabolism ; Perineum ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Sarcoma ; metabolism ; pathology ; surgery ; Vimentin ; metabolism