Objective To investigate the role of ELP_(30)-intein-tag in the prokaryotic expression of recombinant lipoproteinassociated phospholipase A_2 (LP-PL A_2 ). Methods The recombinant plasmid pIG6-ELP_(30)-intein-LP-PL A_2 was transformed into E.coli W3110. Using the recombinant protein LP-PL A_2 without ELP_(30)-intein-tag as negative control,the expression of recombinant ELP_(30)-intein-LP-PL A_2 in periplasm under different temperatures(20,25 and 30 ℃)and induction methods(IPTG and automatic induction)was analyzed by Western blot. The target protein was purified and isolated via inverse transition cycling(ITC)based on elastin-like polypeptide(ELP)-tag,and then detected by SDS-PAGE and Western blot.Results The recombinant LP-PL A_2 protein without ELP_(30)-intein-tag was expressed only intracellularly and not located in the periplasmic space. Under the same induction temperature,the amount of target protein induced by automatic induction was much lower than that induced by IPTG. Compared with 20 ℃,the overall expression level of target protein induced by IPTG increased at 25 ℃ and 30 ℃. The target protein ELP_(30)-intein-LP-PL A_2 was successfully isolated and purified by ITC technique,and the recombinant protein LP-PL A_2 was obtained with the purity of about 70% by inducing intein self-cleavage,which showed specific binding to the anti-LP-PL A_2 antibody. Conclusion The ELP_(30)-intein-tag can promote the expression of recombinant protein LP-PL A_2 in the periplasmic space of E.coli,and the recombinant protein LP-PL A_2 isolated by non-chromatographic purification has high purity,which provides a new method for the rapid,low-cost and effective production of recombinant protein LP-PL A_2 . 关键词(KeyWords)： 脂蛋白相关磷脂酶A_2;类弹性蛋白;原核表达 Lipoprotein-associated phospholipase A_2(LP-PL A_2);Elastin-like polypeptide(ELP);Prokaryotic expression

Objective To develop ECG Holter system with SD card as storage medium. Methods Data that used MSP430F449 SCM to acquire 3-channel ECG signal to record SD card through serial peripheral interface were reviewed and analyzed on computer. Results Portable ECG Holter System based on SD card is realized. The using of SD card can enhance storage performance. Conclusion Low-cost, and high-performance solution program is observed.

Surgical resection and liver transplantation are still the preferred treatments for patients with hepatocellular carcinoma (HCC).For the patients with advanced HCC which are not suitable for surgical resection,traditional chemotherapy can not improve the prognosis.Molecular targeted therapy is a new method and tendency in the treatment of HCC.Multiple targets inhibitors,vascular endothelial growth factor inhibitors and monoclonal antibodies for HCC are widely researched and applied.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; Adult ; Combined Modality Therapy ; Female ; Humans ; Middle Aged ; Moxibustion ; Needles ; Postoperative Complications ; etiology ; physiopathology ; therapy ; Scalp ; Treatment Outcome ; Urinary Retention ; etiology ; physiopathology ; therapy ; Urination ; Uterine Cervical Neoplasms ; surgery

Objective • To construct the E.coli CLM37 strain with Lpp gene deletion and to study the production of N-glycosylated recombinant proteins in this E.coli strain. Methods • Firstly, Red homologous recombination system was used to knock out the Lpp gene from the genome of E.coli CLM37. And then, the growth curve was detected to study the effects of deleted Lpp gene on the growth states of E.coli strain. Finally, the vector pIG6-rFn3-Gly which expresses receptor protein and the vector pACYCpgl, which carries N-glycosylation gene cluster that derives from Campylobacter jejuni, were co-transformed into E.coli CLM37ΔLpp to investigate the extracellular production of N-glycosylated recombinant proteins. Results • The E. coli CLM37ΔLpp with Lpp gene deletion was obtained, and the extracellular production of N-glycosylated rFn3-Gly was successfully achieved in this strain. Compared with E. coli CLM37, the total amount of rFn3-Gly produced via extracellular production of E. coli CLM37ΔLpp increased about 4 times, and the glycosylation efficiency increased about 6 times. Conclusion • N-glycosylated rFn3-Gly was successfully produced via extracellular production in E. coli CLM37ΔLpp, and the production of interest glycoprotein and the glycosylation efficiency were improved.

Through genetic recombination technique, the rat glial cell line-derived neurotrophic factor (rGDNF) cDNA was in-serted into polylinker site of retroviral vector pLXSN, to generate a recombinant plasmid pLXSN-GDNF as transfer vector. Therecombinant plasmid was verified with restriction analysis, PCR, dot blot hybridization and Southern blot hybridization. The re-sults showed that GDNF cDNA was cloned correctly into retroviral vector pLXSN, recombinant retroviral vector was construct-ed. It is concluded that the eukaryotic cell expression vector was constructed successfully for gene therapy of Parkinson's,Alzheimer's and other central nervous system diseases.

In order to investigate effect of basic fibroblast growth factor(bFGF) on the prolifiration and differentiation of theneural progenitor of embryonic hippocampus of rats in vitro, 25 ng/ml bFGF was employed into the serum-free medium of cultureof hippocampal neural cells of embryonic day 18 rats in the present study. The effect of bFGF on the viability of cells culturedwas detected by MTT colorimetric method and the effects of bFGF on proliferation and differentiation of hippocampal neural pro-genitors were analyzed qualitatively and quantitatively by means of immunochemistry, for the nestin, neurofilament, galactocere-broside and glial acidic fibrillary protein. The results showed that the OD value of experimental group was higher than that ofcontrol group by 1.5 and 1.8 times at 4 d and 8 d respectively. The quantitative analysis of each kinds of cells indicated that thenumber of neural progenitors, neurons and oligodendrocytes in experimental group were increased about 2 times as many as thatin control group, but no differences of astrocytes between the two groups at 4 d. However, the number of four kinds of cells aug-mented about 1.7 times at 8 d. The results of this study suggest that bFGF can not only promote the survival, proliferation, butalso facilitate the differentiation of neural progenitor of hippocampus to neurons and glial cells. To obtain more many purifiedneural progenitors in vitro, the embryonic day 18 is not an appropriate age. It is better to get younger embryo brain to culture and to add enough bFGF.

Objective To explore whether the neural stem cells(NSCs) can act directly as a gene target cell which can be infected by the recombinant retrovirus and express the products of exogenous genes after infection. Methods The NSCs were cultured with supernatant containing the recombinant retroviruses with the genes of NGF or GDNF for two days.After screened with G418,the infected NSC were expanded at the present of bFGF in culture.The PC12 cells and the neurons of ventral midbrain of rat were cultured by the medium from the infected NSC,which were called as GDNF\|containing conditioned medium NGF or GDNF\|containing conditioned medium the morphological changes of the dopamine neurons of the ventral midbrain and expression of exogenous genes of the infected NSCs were detected by immunohistochemistry staining. Results It was estimated that about fifty percent of NSCs via retrovirus\|mediated NGF or GDNF gene transduction were G418\|resistant.These infected NSCs began to differentiate.Long and radical processes reached out from the sphere of proliferation and the cells migrated towards outside along the processes.The NSC infected with gene of NGF showed an astroid\|shape with larger body and processes.The NSC infected with gene of GDNF showed a shuttle\|shape with a smaller body and long processes.The PC12 cells increased in the NGF\|containing conditioned medium and stretched out long neurites.The dopamine neuron of the ventral midbrain which were immunoreactive for TH also showed a larger body and longer processes in the GDNF\|containing conditioned medium.Most of G418\|resistant NSCs were immunoreactive for NGF or GDNF. Conclusion NSC can act directly as a gene target cell which not only be infected by the recombinant retrovirus,but also express and secrete the products of exogenous genes.

Objective To investigate the association of serum levels of soluble intercellular cell adhesion molecule-1 (sICAM-1),soluble vascular cell adhesion molecule-1 (sVCAM-1) and high sensitivity C-reactive protein (hs-CRP) with peripheral vascular disease of lower limbs in patients with type 2 diabetes mellitus (T2DM).Methods One hundred and thirty T2DM patients admitted from October 2011 to October 2012,and 30 age/sex-matched healthy subjects were enrolled in the study.The serum levels of sICAM-1,sVCAM-1,hs-CRP and other clinical parameters were measured; the peripheral blood vessels of lower limbs were examined with color Doppler ultrasonography.Based on the extent of angiopathy of lower limbs T2DM patients were classified as normal vascular group (n =26),mild angiopathy group (n =45),moderate/severe angiopathy group (n =59).Results The serum levels of sICAM-1 and sVCAM-1 in moderate/ severe angiopathy group of T2DM patients were higher than those in mild angiopathy group,normal vascular group and healthy controls (t:4.15-8.93,all P ＜0.05) ; the serum levels of hs-CRP in moderate/severe angiopathy group were higher than those in mild angiopathy group,normal vascular group and healthy controls (t:2.18-4.27,all P ＜ 0.05).The serum sICAM-1 level was positively correlated with total cholesterol (TC),low density lipoprotein cholesterol (LDL-C) and sVCAM-1.The serum sVCAM-1 level was positively correlated with course of disease,systolic blood pressure and CRP.Conclusions Serum levels of sICAM-1,sVCAM-1 and hs-CRP are correlated with the extent of angiopathy of lower limbs in T2DM patients,and the elevated sICAM-1 ; sVCAM-1 and hs-CRP levels are also associated with hyper blood pressure,dislipidemia and chronic inflammation.

Objective To investigate the association of serum visfatin and free fatty acid (FFA) with insulin resistance in type 2 diabetes mellitus (T2DM).Methods One hundred and nineteen patients with T2DM and 65 health subjects with normal glucose tolerance (NGT) were enrolled in the study.TheT2DM patients were further classified as insulin resistant (HOMA-IR ＞ 2.8 mU/L,T2DM-IR group,n =61) and non-insulin resistant (HOMA-IR≤2.8 mU/L,T2DM-NIR group,n =58).Serum visfatin,free fatty acid and related clinical variables were measured,and HOMA-IR was calculated.Results The serum levels of visfatin and FFA in T2DM patients were significantly higher than those in healthy controls [(4.7 ±2.5) vs.(1.7±0.9) ng/L,t=-11.831,P＜0.01; (1.65±0.69) vs.(0.61 ±0.21) mmol/L,t=-9.239,P ＜0.01].The serum levels of visfatin and FFA in T2DM-IR group were significantly higher than those in T2DM-NIR group [(6.3±2.3) vs.(3.0±1.4) ng/L,P＜0.01; (2.16±0.45) vs.(1.12± 0.46) mmol/L,P ＜0.01].Multiple regression analysis showed that FFA,fasting insulin level and waist/ hip ratio (WHR) were independent risk factors of serum visfatin level (r =0.564,0.267 and 0.188 respectively,all P ＜ 0.05).Conclusion Serum levels of visfatin and FFA are increased in T2DM,and they are closely associated with insulin resistance.