Tandem expression and purification of tick-borne encephalitis virus E protein Domain Ⅲ and preparation of its polyclonal antibody / 中国生物制品学杂志

@#Objective To express and purify the E protein Domain Ⅲ(ED Ⅲ) of tick-borne encephalitis virus(TBEV) in tandem and prepare the corresponding polyclonal antibody.Methods The TBEV RNA was extracted by Trizol method,and then reversely transcribed into cDNA,which was used as template to amplify ED Ⅲ gene fragment by PCR.Two ED Ⅲ gene fragments were ligated into fusion gene by the hydrophobic flexible polypeptide(G_4S)_3 using overlapping PCR,which was then linked to prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-2ED Ⅲ.After sequencing,pET-28a-2ED Ⅲ was transformed into E.coli BL21(DE3) competent cells,induced by IPTG and purified by Ni~(2+) affinity chromatography.Female New Zealand white rabbits were immunized with the renatured recombinant protein to prepare polyclonal antibody.The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot.The homology of ED Ⅲ amino acid sequence between TBEV and other flaviviruses was analyzed by DNAMAN software.Results The recombinant plasmid pET-28a-2ED Ⅲ was identified by sequencing,and the amplified sequence contained two genes consistent with the E sequence of TBEV "Senzhang" strain(JQ650523.1) included on GenBank,indicating that the recombinant plasmid was constructed correctly.The recombinant 2ED Ⅲ protein was expressed mainly in the form of inclusion bodies,with a relative molecular mass of about 21 000 and a purity of 97.5%.The titer of rabbit anti-2ED Ⅲ serum polyclonal antibody was 1:10~7,which reacted specifically with TBEV whole virus.DNAMAN software alignment showed that the homology of ED Ⅲ amino acid sequences between TBEV and Japanese encephalitis virus(JEV),yellow fever virus(YFV) and Dengue virus(DENY) was 36.56%,9.28% and 30.77%,respectively.Conclusion The TBEV envelope ED Ⅲ tandem recombinant expression plasmid pET-28a-2ED Ⅲ was successfully constructed.The expressed recombinant 2ED Ⅲ protein had good reactivity and immunogenicity,and the prepared polyclonal antibody had high titer.

Structure-based pharmacophore design and screening for potential inhibitors of E protein of dengue virus as a loom for dengue

Dengue virus (DENV) poses a continuous threat worldwide with an estimated 2.5 billion people at the risk of dengueinfection. It was believed to be the infection of the tropical regions, but reports of dengue infection have now extendedand spread around the globe. The dengue E protein is involved in the viral fusion and could thus acts a potentialtarget against dengue virus. In the present study, structure-based pharmacophore design and screening and absorption,distribution, metabolism, excretion, and toxicity (ADMET) analysis using Discovery studio (4.0) was applied toidentify potential hits against the hydrophobic pocket of dengue E protein. The pharmacophore feature of screenedcompounds was further validated and finally three lead compounds were obtained. The pharmacophore model andthe docking study were generated three lead molecules Ophiopoginin D with a binding energy of −146.36 Kcal/mol followed by Calmisttrin D with a binding energy of −118.73 Kcal/mol and BTB 08305 with a binding energy of−99.96 Kcal/mol and exhibited best-fit value. ADMET profile showed that all the three lead molecules are non-toxic,non-carcinogenic, and non-hepatotoxic by in silico study. The compound Calmisttrin D exhibited good blood brainbarrier permeability and human intestinal adsorption, and thus hypothesized to have antiviral activity against denguevirus and so further in vitro and in vivo evaluation is recommended.

A Computational Approach On Understanding Structural Interactions Of Envelope Protein Of Dengue Virus Bound With Squalene, A Prototype Anti-Viral Compound

Objective: The objective of the work was to validate the structural binding affinity of Squalene with the envelope protein of Dengue virus by means of molecular simulations. Methods: Three-dimensional (3D) structure of dengue 2 virus envelope protein was retrieved from Protein Data Bank PDB and Squalene compound from the ZINC database. Molecular docking between the E protein and Squalene were carried out by means of Auto Dock 4.2. Results: Based on the study, it was observed that the binding/docking energy for the complex structure was calculated to be-5.55 kcal/mol. Critical residues to interact with E protein were scrutinized by analyzing the interface of the complex within 4 Å proximity. Residues such as Thr 48, Glu49, Ala 50, Val 130, Leu 135, Ser 186, Pro 187, Thr 189, Gly 190, Leu 191, Phe 193, Leu 198, Leu 207, Thr 268, Phe 279, Thr 280, Gly 281, His 282 and Leu 283 were found to be non-covalently located around the squalene. Conclusion: Scopes to design de novo anti-viral compounds to the dengue viruses by using squalene as a new class of template structure have also been concisely brought into fore.

Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

BACKGROUND: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). METHODS: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-gamma staining. RESULTS: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. CONCLUSION: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2

BACKGROUND: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). METHODS: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-gamma staining. RESULTS: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. CONCLUSION: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein- specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

Prediction of HLA-A~*0201-restricted CTL epitopes in SARS-CoV E protein / 第三军医大学学报

Objective To predict the HLA A *0201 restricted CTL epitopes (nonamers) in SARS coronavirus (SARS CoV) E protein. Methods The CTL epitopes of E protein were predicted by using supermotif method combined with quantitative matrix method. Results Thirteen HLA A *0201 restricted CTL epitope candidates were predicted in SARS CoV E protein. Conclusion The prediction of the CTL epitopes in SARS CoV E protein will benefit the identification of CTL epitopes by experiment. Those results are of importance for immune recognition and vaccine design for SARS CoV.

Gene cloning and expression of Japanese encephalitis virus E protein in Escherichia coli / 中华传染病杂志

Objective To express Japanese encephalitis virus (JEV) E protein in Escherichia coli. Methods The gene coding for JEV E protein was amplified by using RT-PCR, and cloned into plasmid pET-28a. The constructed plasmid was transformed into E.coli BL21 (DE3). Expression of E protein by the transformants was induced by IPTG and analyzed by SDS-PAGE and Western blot. Results Five nucleotide changes leading to amino substitutions were identify in the E protein gene of our laboratory strain of JEV when compared to a published gene sequence of JEV E protein. The yield of expressed JEV E protein with relative molecular mass approximately 52?10 3 was 35% of total bacterial proteins. Conclusions JEV E protein was expressed successfully in E.coli, which should be useful for the production of diagnostic reagents and the analysis of gene structure/function of the E protein.

An experimental study of gene vaccines pcDNA3-pac and pcDNA3-gtfB of Strept ococcus mutans against dental caries in rats:the influence on weight / 实用口腔医学杂志

0.05);by the end of the experiment that was 24.5?1.05,28. 3?1.29,26.6?1.19,10.2?1.81, 26.3?1.54 and 27.3?1.38 respectively (D vs each of the other groups P0.05). Conclusion: Gene vaccines pcDNA3-pac and pcDNA3-gtfB have no unfavo rable influence on weight of gnotobiotic rats,while the inactive whole cell vac cine has.

Expression and clinical significance of tGF-?1,Cyclin E in gallbladder carcinoma / 中国癌症杂志

Purpose:To investigate the expression of TGF-?1,Cyclin E in (primary) gallbladder carcinoma and its clinical significance. Methods:The expression of TGF-?1,Cyclin E in gallbladder carcinoma was detected by S-P immunohistochemical staining,20 cases of chronic cholecystitis were collected as control. Results:①The positive rate of TGF-?1(63.89%),Cyclin E(47.22%) in gallbladder carcinoma increased significantly(P

Effect of Naoxintong Capsule on the Carotid Plaque and Serum Hypersensit ive C-reactive Protein of the Patients with Acute Coronary Syndrome / 中医杂志

Objective To study the functional mechanism of Naoxin tong Capsule (Ca psule for cerebral and heart diseases) on a cute coronary syndrome (ACS). Methods Totally 105 ACS p atients diagnosed with coro nary angiography were randomized into a treatment group (55 cases), and a control group (50 cases). The control group was treated routin ely with western medicine while the treatment group treated with Naoxintong Capsule, 2 capsules each time, 3 times a day, in addition to the routine western medicine. Before and 4 weeks after treatment, the serum hypersensitive C-react ive protein (hs-CRP) was measured. Before and six months af ter treatment, changes of carotid intima-media thickness (IMT) and carot id plaque were examined with Color Doppler Ultrasonography. Resu lts Four weeks after treatment, the hs-CRP level in the treatment grou p was significantly lowered than before treatmen t, and the effect was more significant than that of the control group (P