Objective To construct, express and identify the anti-idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT-PCR method, the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly_4ser)_3 linker, and the scFv in the orientation of V_L-linker-V_H was constructed. It was then cloned into vector plasmid pET-28a, expressed in Escherichia coli BL21(DE3), and confirmed by SDS-PAGE, Western blot and ELISA. Results The recombinant scFv could be expressed in E.coli BL21 (DE3) in a fusion protein pattern. The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded. The SDS-PAGE and Western blot analysis showed that the molecular had the binding activity to the antigen. Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21 (DE3), providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.

Objective To construct,express and identify the anti-idiotypic antibody single chain variable fragment(scFv) against Edwardsiella tarda.Methods By using RT-PCR method,the variable regions of the heavy and light chain of the anti-idiotypic monoclonal antibody(mAb) 1E11 against Edwardsiella tarda were cloned and joined with a(Gly4Ser)3 linker,and the scFv in the orientation of VL-linker-VH was constructed.It was then cloned into vector plasmid pET-28a,expressed in Escherichia coli BL21(DE3),and confirmed by SDS-PAGE,Western blot and ELISA.Results The recombinant scFv could be expressed in E.coli BL21(DE3) in a fusion protein pattern.The expression product was in the form of an inclusion body and the purified fusion protein was obtained after being purified and refolded.The SDS-PAGE and Western blot analysis showed that the molecular weight of scFv protein was 27 ku.Indirect ELISA confirmed that the scFv had the binding activity to the antigen.Conclusion The recombinant anti-idiotype scFv has been successfully constructed and expressed in E.coli BL21(DE3),providing the basis and potential for preparation of genetically engineered vaccine against Edwardsiella tarda.

AIM:To discuss the effect of Shenmai injection on insulin resistance ( IR) in 3T3-L1 cells and its mechanisms.METHODS:3T3-L1 preadipocytes were induced by chemical reagents to differentiate into fully differentiated adipocytes.Oil red O staining was used to detect the differentiation level of the adipocytes .The insulin-resistant 3T3-L1 cell model was demonstrated using insulin , which was confirmed by glucose concentration in cell supernatant .The IR cell model was given 10 μmol/L rosiglitazone , 25 and 50 g/L Shenmai injection and normal saline for comparison .MTT assay was used to assess the cell activity of 3T3-L1 cells which was treated with drugs for 8, 16, 24 and 36 h.Glucose oxidase method was used to detect the glucose concentration in the cell supernatant at 8, 16 and 24 h.The protein levels of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), AKT and p-AKT were determined by Western blot .RE-SULTS:3T3-L1 adipocytes were successfully induced as shown by the positive oil red O staining .The IR cell model was demonstrated , and glucose concentration in the cell supernatant after treatment with Shenmai injection showed that Shenmai injection reduced the IR in 3T3-L1 cell model.The protein levels of GLUT4, PI3K and p-AKT increased compared to con-trol group.CONCLUSION:Shenmai injection reduces the IR in 3T3-L1 cell model, which functions by increasing the protein levels of GLUT4, PI3K and p-AKT.

OBJECTIVETo study the effect of human calcyclin binding protein (CacyBP) encoding gene on the development of multiple drug resistance in gastric cancer.

METHODShCacyBP sense nucleic acid eukaryotic expression vector (pcDNA3.1/hCacyBP +) was constructed and then transfected steadily into the gastric cancer drug sensitive cell (SGC7901) mediated by lipofectamine ( trade mark ) 2000. RT-PCR was used to measure the CacyBP mRNA expression level. MTT was used to measure the adriamycin (ADR) drug sensitivity of SGC7901 and SGC7901 after transfection. FCM was used to measure the average ADR accumulation concentration and cell cycle of SGC7901 and SGC7901 after transfection.

RESULTSThe hCacyBP mRNA expression level of SGC7901 transfected with pcDNA3.1/hCacyBP + was higher than SGC7901 transfected with pcDNA3.1 or SGC7901, with the higher survival rate in the former. The average ADR accumulation concentration in SGC7901 and SGC7901 transfected with pcDNA3.1 or pcDNA3.1/hCacyBP + was 5.64, 5.49 and 5.17, respectively. The G(1) phase cell proportion of SGC7901 transfected with pcDNA3.1/hCacyBP + or pcDNA3.1 was reduced slightly but G(2) and S phases increased slightly as compared with SGC7901.

CONCLUSIONCalcyclin binding protein may play a certain role in gastric cancer drug resistance.

Base Sequence ; Calcium-Binding Proteins ; genetics ; physiology ; DNA, Complementary ; analysis ; Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Drug Screening Assays, Antitumor ; Gene Transfer Techniques ; Humans ; Molecular Sequence Data ; Plant Lectins ; Stomach Neoplasms ; pathology ; Tumor Cells, Cultured

Objective To explore the value of anti-Müllerian hormone (AMH) and age in predicting outcomes of patients undergoing in vitro fertilization and embryo transfer treatment. Methods In this retrospective study, 6 328 Chinese patients who underwent the first in vitro fertilization or intracytoplasmic sperm injection and embryo transfer treatment in Henan Provincial People′s Hospital between July 2016 and July 2018 were analyzed. All the patients were categorized into two groups according to pregnancy or not. Baseline data and outcomes of two groups were compared. The regression analysis was conducted to identify the independent factors of clinical pregnancy rates. Furthermore, correlation analysis was performed between AMH and other factors. Results (1) The total clinical pregnancy rate was 56.86% (3 547/6 238). Age, AMH, basal FSH, antral follicle number (AFC), starting dose of gonadotropin (Gn), total doses of Gn, duration of Gn, number of oocytes, transferable cleavage embryos and transferred embryos were significantly different (all P<0.01). (2) Correlation analysis showed that AMH had significant passive correlation with age, basal FSH, starting dose of Gn and total doses of Gn (all P<0.01), while showed significant positive correlation with AFC, body mass index, duration of Gn, number of oocytes and transferable cleavage embryos (all P<0.01). Of all the factors, AMH had the strongest correlation with AFC (P<0.01). (3) Multivariate logistic regression analysis suggested that age was the independent influencing factor of clinical pregnancy rate ( OR=0.938, 95%CI : 0.824-0.952, P<0.01), while AMH not ( OR=1.004, 95%CI : 0.984-1.024, P=0.687). In the subgroups according to age, the advanced group (age>35 years old) had lower clinical pregnancy rate and higher cancellation rate for no available embryos. Conclusions AMH has no predictive value of clinical pregnancy outcomes for patients with in vitro fertilization and embryo transfer treatment, while age has certain predictive value of pregnancy outcomes. AMH level may have indictive value for the evaluation of ovarian reserve.

Objective To investigate high risk human papillomavirus(HR-HPV)prevalence among married women in Beijing and to study the high risk flactors.nethods During March 2007 to September 2008.a total of 6185 married women sampled from 137 communities in 12 districts were screened bv HR-HPV DNA test and cytogical test.The interview was carried out with unified questionnaires.The databage was set up and twice entered in EpiDam 3.0.After checked up,the data were analyzed in SPSS 15.0.Results (1) The HR-HPV infection rate was 9.89%.The HR-HPV infection rate of the city zone,the suburb and the exurb were 9.34%,10.51% and 9.51% (P>0.05).The HR-HPV infection rate of the native and the oudander were 9.53%,11.30% (P<0.05).(2) The age distribution of HR-HPV infection was that the rate was around 10% among 25 to 44 age groups,which was the highest(11.21%) in 30 to 34 age group;then the rate was descended as the age raising,the rate of 50 to 54 age group was the lowest(7.78%).(3) Multiple logistic regression showed that the related risk factors of HR-HPV infection mainly included 1000 RMB and above of family income per person per month.possessing more than 1 sexual partner of her husband,outlander and hish levels of education.(4) The prevalence of cervical intraepithelial neoplasia(CIN)in HR-HPV positive group wag significantly higher than that in HR-HPV negative group(29.76% vs 3.32%,P<0.01).Conclusions(1)The HR-HPV infection rate among aged 25 to 54 years was 9.9% and there was no significant difference in area distribution.(2)The hish risk population which should strengthen screening was the married bearing-age women with high level of family income,outlander,high levels of education and her husband possessing more than 1 sexual partner.(3)HR-HPV infection is the main risk factor for CIN and cervical cancer.while does not provide a causal relationship with them.The high risk population should be checked regularly to understand the development of HR-HPV infection and CIN incidence.