Objective To express lactate dehydrogenase (LDH) gene of Plasmodium falciparum FCC1/HN in the E. coli TG1 and analyse its immunocompetence. Methods The LDH gene of the P. falciparum was specifically amplified by polymerase chain reaction, and the recovered gene fragment was cloned into pGEX 4T 1 vector for expression of fusion protein with glutathione S transferase(GST). The recombinant plasmid was transformed into the E. coli TG1. Four mice (Kunming strain) were immunized with purified expressed protein(antigen) and the polyclonal antibodies were collected. The immunocompetence of recombinant protein was analysed by ELISA and Western blot. Results The LDH gene of P. falciparum was successfully expressed in the E. coli TG1. The expressed protein exhibited a specific reaction with immune sera obtained from rabbits immunized with P. falciparum . The specific humoral responses were induced in mice and the titer of the specific antibody was 1∶16 by two dimensional diffusion assay. Conclusion The LDH gene of P. falciparum has been successfully expressed in the E. coli TG1 and the expressed protein has high antigencity.

100 parasites/?l, 72.73% with

Efficient gene delivery and sustained gene expression are required for successful human gene therapy.Although viral vectors are considered the most efficient vehicles for gene transfer,currently available viral vectors have not fully achieved these two requirements.Lentiviral vectors(LVs)can integrate into host chromosomes,allowing long-term gene expression,in addition,these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome,but are still limited to in vitro or ex vivo gene delivery because of their relatively low titers using transient transfection experiments.In order to develop an efficient transient transfection method for large-scale production of high titer lentiviral vector stocks,a minimal lentiviral vector producing system based on vaccinia virus that synthesizes T7 RNA polymerase was developed.BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA,the packaging plasmid pGAGPOL and the envelope plasmid pVSVG,and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000TM.After 4 days,the culture supernatant of lentiviral vectors was collected,the RNA from the supernatant was examined by the RT-PCR,the protein from the supernatant was examined by Western blot,and the supernatant was used to transfect normal 293T,HepG2 and Vero,which were observed by the immunofluorescence microscopy.The type of cell lines,plasmids dosage and the MOI(the proportion between cell numbers and virus copies)were considered so critical to the output of this system that 3?3?3 factorial design was used to explore the yield optimization of this system.As judged by the results of RT-PCR and the Western blot,lentiviral vectors were found in the culture supernatant;as judged by immunofluorescence with microscopy,293T,HepG2 and Vero which were transfected by the supnantant expressed the report protein-green fluorescent protein(GFP),the results confirmed the valid infectivity of the lentiviral vector produced by the system.Eventually,the best titers of lentiviral vector stocks was up to 1.3?108 tu/ml,which is one order of magnitude higher than the output of classical manufacture system.The new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established.It provides the basis for the future development of industrial application.

Objective To prepare the human dual-specificity protein phosphatase18 (Dusp18) monoclonal antibodies (McAb) with high titer and specificity and identify its characterization, which is based on further studying Dusp18 function. Methods BALB/c mice were immunized with purified recombinant Dusp18 protein. Cell fusion was performed between mouse splenic cells and myeloma cells (SP2/0), and then the hybridoma cell lines secreting McAb against Dusp18 antigen were screened and cloned. The ascites were prepared and purified with Protein G affinity chromatography. The titer and subtypes of McAb against Dusp18 were identified and measured by ELISA and Western blotting analysis. Results Two hybridoma cell lines, F003 and F004, that stably secreted McAb against Dusp18 were successfully obtained, which belong to the subtypes of IgG1 and k light chain. The antibody titers in culture supematant were 1:5120 and1:10 240, and those in the ascites fluid were 1:25 600 and 1:51 200 respectively. Western blotting analysis and immunohistochemistry showed that the two antibodies can specifically bind with Dusp18 derived from human eucaryotic cells or tissue. Conclusion Two McAb against Dusp18 have been successfully prepared which can be used for further studying the biological properties of Dusp18 and reveal its relationship with tumorigenesis and development.

Objective To observe the effect of clindamycin for preventive infection in arthroplasty prophylac-tic.Methods 108 knee replacement patients were injected by 600mg intravenous clindamycin preoperation and con-tinue to use 1 -2 days after operation.The average postoperative hospitalization,postoperative outcome,body tempera-ture and blood after WBC changes of CRP and ESR in the fall of superficial infection trend,the average postoperative day,take out stitches after operation and the number of cases,postoperative deep infection early after infection (2 weeks)and delayed infection cases (within one year)were observed in order to evaluate the efficacy of preopera-tive antibiotics.Results 2 cases had superficial infection due to wound dehiscence (two times after suture recover-y),there was no complications of surgical wound in the other cases.In all cases,after operation,body temperature and blood WBC became to the normal level in seventh days,postoperative patients'CRP,ESR monitoring were significantly higher than those before operation (CRP:F =105.32,P =0.045;ESR:F =118.47,P =0.039),but on the 5 day after operation they were started to decline,CRP in the 21 postoperative day gradually returned to normal,and ESR gradually returned to normal after 6 months of operation.Preoperative HSS score was significantly lower than the post-operative score[(46.8 ±9.7)points vs.(91.7 ±3.4)points,t =6.38,P <0.05].Conclusion Clindamycin plays a definite role in prevention of infection,especially in the beta lactam antibiotic allergy cases,it can be preoperative antibiotic prophylaxis instead of cephalosporins.

Objective To investigate the serum anti-HBc level in patients with different natural history of chronic hepatitis B virus (HBV) infection and cirrhosis,and its clinical value for distinguishing the natural history statue.Methods A total of 160 patients with chronic HBV infection from March 2015 to June 2016 were enrolled,and they were divided into immune tolerance group (n=43),HBeAg-positive chronic hepatitis B (CHB) group (n=37),inactive carrier group (n=39) and HBeAg-negative CHB group (n=41).A total of 44 patients with HBeAg-positive cirrhosis and 46 patients with HBeAg-negative cirrhosis were enrolled too.The general conditions data were collected,and HBsAg,HBeAg,anti-HBc,HBV DNA load and HBV genotype were detected.The associations between anti-HBc level and clinical parameters were analyzed,and the diagnostic value of anti-HBc for distinguishing different natural histories was analyzed.Results The anti-HBc levels in different natural history from high to low were as following: HBeAg-positive CHB group (4.22±0.68)log10 IU/mL,HBeAg-negative CHB group (3.89±0.88)log10 IU/mL,inactive carrier group (3.07±0.68)log10 IU/mL and immune tolerance group (2.88±0.82)log10 IU/mL.The anti-HBc levels in HBeAg-positive and HBeAg-negative cirrhosis patients were (3.04±0.82) and (3.15±0.86) log10 IU/mL,respectively.In HBeAg-positive CHB group,the anti-HBc was positively associated with ALT (r=0.353,P=0.032) and AST (r=0.421,P=0.009).In HBeAg-negative CHB group,the anti-HBc was positively associated with HBV DNA (r=0.343,P=0.028),ALT (r=0.458,P=0.003) and AST (r=0.495,P=0.001).The AUC of anti-HBc used to distinguish immune tolerance from HBeAg-positive CHB was 0.903,and the AUC used to distinguish inactive carrier from HBeAg-negative CHB was 0.833.Conclusion Anti-HBc levels in different natural history of chronic HBV infection are significantly different,and anti-HBc could be used to distinguish the natural history statue of chronic HBV infection with a higher diagnostic value than HBsAg.

Objective: To better understand homing potentiality of hematopoietic stem cells (HSC) in human umbilial cord blood (UCB) and the mechanism of dextran sulfate (DS) mobilization. Methods: Sub lethally irradiated or DS pretreated severe combined immunodeficient (SCID) mice were transplanted with UCB, which was cryopreserved at -80℃.Human cells in recipient mice were detected by flow cytometry and CFU GM assay from each host organ. Results: In contrast with the controls, engraftment after irradiation or administration of DS resulted in a higher percentage of CD45 +,CD34 +,CD19 + cells produced in SCID mice. While comparison between the experimental groups, higher implantation level was obtained in the former if equivalent donor cells were used in both groups. Conclusion:DS is a safe and effective pretreatment, which can mobilize HSC, but also vocate niches for transplanted HSC homing. [

Objective:To establish a colloidal gold technique assay for the rapid detection of immunoglobulin(Ig)M and IgG antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance.Methods:A total of 278 patients who were respectively treated at Wuhan Hankou Hospital and the People′s Hospital of Honghu from February 12, 2020 to February 20, 2020 were collected. According to the diagnostic criteria, 89 patients were confirmed with positive 2019-nCoV nucleic acid, and 189 were 2019-nCoV nucleic acid-negative suspected patients. A total of 273 medical examiners from Nanfang Hospital, Southern Medical University from 2015 to 2018 were selected as controls. The serum samples of patients were collected. 2019-nCoV nucleic proteins were obtained from prokaryotic expression vectors. Indirect IgM and IgG colloidal gold techniques were established by using recombinant nuclear protein. 2019-nCoV nucleic acid detection by reverse transcription-polymerase chain reaction (RT-PCR) was used as control. Serum specimens were tested for 2019-nCoV IgM and IgG. The specificity and sensitivity of colloidal gold assay were analyzed.Results:The positive rates of IgM and IgG with the colloidal gold detection in confirmed patients with positive 2019-nCoV nucleic acid were 78.7%(70/89) and 73.0%(65/89), respectively. The positive rates of IgM and IgG in medical examiners were 1.8%(5/273) and 0.7%(2/273), respectively. The sensitivity and specificity of IgM detection reagents were 78.7% and 98.2%, respectively, those of IgG detection reagents were 73.0% and 99.3%, respectively, and those of IgM combined with IgG detection were 87.6% and 98.2%, respectively. For suspected patients with negative 2019-nCoV nucleic acid, the positive rates of IgM and IgG were 59.8%(113/189) and 52.9%(100/189), respectively, and the positive rate of IgM combined with IgG detection was 66.1%(125/189).Conclusion:This reagent of 2019-nCoV antibodies detection (colloidal gold technique) fulfills the requirement for clinical application with high specificity and sensitivity, which can be served as a supplementary detection method for 2019-nCoV nucleic acid detection by RT-PCR.

Objective: To establish a colloidal gold technique assay for the rapid detection of immunoglobulin(Ig) M and IgG antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance. Methods: A total of 278 patients who were treated at Wuhan Hankou Hospital and the People's Hospital of Honghu from February 12, 2020 to February 20, 2020 were collected. According to the diagnostic criteria, 89 patients were confirmed with 2019-nCoV nucleic acid positive diagnosis, and 189 were 2019-nCoV nucleic acid-negative suspected patients. A total of 273 medical examiners from Nanfang Hospital, Southern Medical University from 2015 to 2018 were selected as controls. The serum samples of patients were collected. 2019-nCoV nucleic proteins were obtained from prokaryotic expression vectors. Indirect IgM and IgG colloidal gold techniques were established by using recombinant N protein. 2019-nCoV nucleic acid detection by reverse transcription-polymerase chain reaction (RT-PCR) was used as control. Serum specimens were tested for 2019-nCoV IgM and IgG. The specificity and sensitivity of colloidal gold assay were analyzed. Results: The sensitivity and specificity of IgM detection reagents were 78.7% and 98.2%, respectively, those of IgG detection reagents were 73.0% and 99.3%, respectively, and those of IgM combined with IgG detection were 87.6% and 98.2%, respectively. For suspected patients with negative 2019-nCoV nucleic acid, the positive rates of IgM and IgG were 59.8% (113/189) and 52.9% (100/189), respectively, and the positive rate of IgM combined with IgG detection was 66.1% (125/189). Conclusion This reagent of 2019-nCoV antibodies detection (colloidal gold technique) fulfills the requirement for clinical application with high specificity and sensitivity, which can be served as a supplementary detection method for 2019-nCoV nucleic acid detection by RT-PCR.