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This article reviews the research advances in large, middle, and small hepatitis B virus (HBV) surface proteins, including their gene structures, characteristics, detection methods, and clinical significance. Up to now, the clinical significance of large, middle, and small HBV surface proteins remains unclear and requires further studies.
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The pre-S/S gene of hepatitis B virus (HBV) can encode for the production of large, medium and small surface protein. Different protein expression levels and their composition ratios have certain influences on the diagnosis, treatment and outcome of HBV infection. It is of great significance to clarify the functions of large, medium and small surface protein as serum markers and to explore their value in the diagnosis and treatment of HBV infection. In this paper, the expression status, detection methods and clinical significance of the three HBV proteins were reviewed.
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West Nile virus(WNV)is a member of the Flavivirus genus, which can cause zoonotic diseases or even death. With the gradual globalization of WNV infection epidemic, there exists an input risk in China. Now there are no approved vaccines and drugs for the prevention and treatment of human infection with WNV. Hence, research and de- velopment of drugs against WNV is necessary. WNV envelope(E)protein, the main antigen of neutralizing antibody, is involving in the infection of the target cells by the virus. The E protein contains three structural domains(D, DⅡ and DIII). This review briefly describes the recent advances in neutralizing antibodies targeting these three domains.
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Objective To investigate the expression of human papilloma virus L1 (HPV L1)capsid protein in cervical lesions and different human papillomavirus ( HPV) subtypes, and to guide clinical triage management and best individual treatment.Methods Retrospective analysis of 2012 January to 2014 Janu-ary in Jiaxing Hospital of Traditional Chinese Medicine gynecology clinic for HPV L1 protein combined with HPV type, and liquid-based cytology test ( TCT) of 176 patients data.Results The positive expression rate of HPV L1 protein with TCT examination in the negative for intraepithelial lesion or malignancy ( NILM) , atypical squamous cells of undetermined significance( ASCUS) , low-grade squamous intraepithe lial lesion ( LSIL) , atypical squamous cells not except high lesion ( ASC-H) , high-grade squamous intraep-ithelial lesion ( HSIL) , and squamous-cell carcinoma ( SCC) was 28.99%, 44.19%, 64.44%, 22.22%, 12.50%, and 0, respectively.No significant differences were found between the NILM and ASCUS groups ( P >0.05) .The positive expression rate of HPV L1 protein in LSIL group was the highest, and it was sta-tistically significantly different from ASC-H and HSIL groups (χ2 =3.88,5.50, P 0.05 ); and statistically significant difference was found between CIN Ⅰgroup and CINⅡ, CINⅢgroup (χ2 =4.53,5.56, P 0.05) .Conclusions Detection of HPV L1 protein is of clinical value to evaluate the risk of cervical lesions.HPV L1 protein combined with HPV type and TCT detection is helpful for traffic man-agement and personalized treatment, and benefit patients with cervical lesions.
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Objective To study the evolutionary and mutant characteristics of Ebora virus envelope glycoprotein.Methods A total of 100 Ebora virus envelope glycoproteins amino acid sequences isolated during 1976 and 2014 were collected from National Center for Biotechnology Information (NCBI).Multiple sequence alignment and phylogenetic tree analysis were performed to investigate the evolutionary and mutant characteristics of Ebora virus envelope glycoprotein.Results Glycoprotein amino acid sequences of Ebora virus isolated during 1976 and 2014 showed only 54.00％-65.00％ homology among different subtypes,while 95.00％-100.00％ homology in same subtypes.Ebola virus isolated from different regions in 2014 showed a 99.70％-100.00％ homology of glycoprotein amino acid sequences in the same subtype.The homology of glycoprotein amino acid sequences of Ebola-Zaire virus isolated from Sierra Leone in 2014 was 100.00％,but three strains of Ebola-Zaire virus isolated from Guinea showed diversity in glycoprotein amino acid sequences.Glycoprotein amino acid sequences of Ebola virus with different subtypes were on different branches of phylogenetic tree.Glycoprotein amino acid sequences of Ebola-Zaire virus isolated from Sierra Leone in 2014 were on one branch,and those of Ebola-Zaire virus isolated from other countries during 1976 and 2014 were on the another branch.Conclusions Glycoprotein amino acid sequences of Ebora virus vary with time and region.Ebola-Zaire virus isolated from different regions in 2014 may be two variants with the same origin,and hybrid phenomenon is not observed among virus of different subtypes.
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Objective To establish a model for preparation of tissue-engineered skin grafts with hTERT cells carrying human papillomavirus type 6 (HPV 6) genome in vitro, so as to lay a foundation for studying HPV life cycle. Methods The full-length linear HPV6 genome and plasmid pEGFP-▲EGFP were electrophoretically cotransferred into hTERT cells. After selection using G418 resistance, Southern blotting was performed to determine the viral load of HPV6 in transfected cells. 3T3 J2 trophoblastic cells, type I rat-tail collagen and hTERT cells containing the full-length HPV6 genes (HPV6.hTERT cells)were mixed and cocultured on metal meshes to form skin graft-like structures. Hematoxylin and eosin (HE)staining was performed to observe the structure of formed skin grafts, an immunohistochemical assay to measure the expression of HPV6 L1 protein, and electron microscopy to observe virus particles in the skin grafts. Results The linear HPV6 gene was successfully transferred into hTERT cells, and Southern blotting showed the presence of HPV6 DNA in the transferred hTERT cells. The HPV6.hTERT cells, which were cocultured with 3T3 J2 trophoblastic cells and type I rat-tail collagen, proliferated and differentiated over time, and gradually formed skin grafts giving the appearance of verrucous hyperplasia. HE staining showed that the cocultured HPV6.hTERT cells could form typical stratified structure of skin after 7 days of cultivation, and histopathologic features of HPV infection, including obvious papillomatous hyperplasia, presence of vesicular cells, hyperkeratosis and parakeratosis, could be observed after 21 days. The immunohistochemical assay showed the expression of HPV6 L1 protein in the upper portion of skin grafts, and electron microscopy revealed the presence of HPV6 virus particles in skin grafts. Conclusions The established model for preparation of tissue-engineered skin grafts using HPV 6 genome-carrying cells provides a basis for biological studies of HPV, but its application is limited to some degree.
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Objective To establish a method of TRFIA with high sensitivity and broad detecting range for serum HBV large surface protein (HBV-LP).Methods The monoclonal antibody of HBV-LP was covered on the microwell plate and incubated with HBV-LP in blood sample,then Eu3+ labeled antibody of HBs was added.HBV-LP in standard substance,blood samples of 66 chronic hepatitis B patients and 30 healthy controls was detected by the TRFIA and ELISA.x2 test and linear correlation analysis were used for data analysis.Results The dose-response curve of standard substance with TRFIA had good linear correlation (r=0.999).Normal reference range was established at 0-1.36 mg/L based on the ELISA results of 30 healthy controls.The sensitivity was 0.10 mg/L.The specificity was 100％ (30/30).Correlation coefficient between the TRFIA and the ELISA was 0.800 9 (P＜0.001).The positive detecting rates of the 2 methods were significantly different (89.4％(59/66) vs 77.3％(51/66),x2 =6.13,P＜0.01).The recovery rate for HBV-LP was between 95.93％-107.62％.The effective detecting range(CV＜10％) of TRFIA was 1.35-2 764.00 mg/L,and that of ELISA was 10.8-691.0 mg/L.Conclusion The TRFIA was established for HBV-LP detection with higher sensitivity and wider detecting range compared to ELISA.It has potential value for HBV screening and monitoring of antiviral therapy.
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ObjectiveTo analyze the Envelope (E) gene of type 1,2,3 dengue virus isolated fromGuangzhouin2010, andtoinvestigatetheinfectionsourceandvirusgenotypes.MethodsEighty-five serum samples were collected from 85 patients in acute phase of dengue fever.Dengue virus was cultured and isolated by C6/36 cells.The whole length of E gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and then sequenced.The phylogenetic tree was drawn by neighbor-joining method.The bioinformatics analysis was performed by combining the phylogenetic information and the epidemiologic data.ResultsSix strains of type 1 dengue virus,two strains of type 2 dengue virus and six strains of type 3 dengue virus were isolated from 85 samples.The E gene sequence of these strains was obtained by sequencing.The phylogenetic analysis showed that type 1 and 3 dengue virus belonged to two genotypes (Asian and South Pacific ocean,India subcontinent and Southeast Asia/South Pacific ocean,respectively),and type 2 dengue virus belonged to one genotype (Malaysia/India subcontinent).ConclusionIt's presumed that all strains of type 2 dengue virus are imported,four strains of type 1 dengue virus are imported and four strains of type 3 dengue virus arc imported,the remaining two stains of type 1 and two stains of type 3 dengue virus need mosquito intermediary research further to prove their origins.
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Objective To explore the differential liver plasma membrane (PM) proteins that may be related to the occurrence,development and reversal process of hepatitis and to understand the pathogenesis of hepatitis and the new drug targets by performing a comparative proteomics research of liver PM between hepatitis B surface antigen (HBsAg) transgenic mice and wild-type C57 mice.Methods A 6-month-old HBsAg transgenic mouse model was established.The pathological examination was performed to observe the pathological changes of transgenic mice and wild-type C57 mice.The PM from liver tissue of 6-month-old transgenic mouse and the control mouse were purified through twice sucrose density grade centrifugation combined with second antibody magnetic bead enrichment.The purity of extracted PM was verified by Western blot.Differential proteome expression analysis was performed by using two-dimensional electrophoresis (2-DE) and ImageMaster software analysis.The differentially expressed proteins were lysed by trypsin and identified by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis.Results The pathological examination results showed that hepatitis was observed in the transgenic mouse group,while no abnormity was found in the controls.The PM was successfully enriched and the mitochondria contamination was reduced by sucrose density grade centrifugation combined with second antibody magnetic bead purification treatment.Thirty differential mice liver PM protein spots were visualized,in which 11 non-redundant proteins were successfully identified by LC-MS/MS in transgenic mouse group,including 9 up regulated protein spots and 2 down-regulated protein spots.These differentially expressed proteins included keratin,cardiac Ca2+ release channel,cytochrome B5,ATP synthase subunit alpha,etc.Conclusions A batch of HBsAg gene expression related differential proteins are identified in mouse liver plasma.These proteins might be new drug targets for anti-HBV treatment.This study will guide further investigation on the mechanism of HBV infection induced hepatitis.
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Objective To investigate the characteristics of V3 loop amino acid sequences of human immunodeficiency virus type 1 (HIV-1) quasi-species in long-term non-progressors (LTNP)infected with HIV. Methods End-point limiting dilution polymerase chain reaction (EPLD PCR) was used to amplify the env gene c2-v3-c3 region of single HIV-1 provirus from five LTNPs at sequential time points. The PCR products were then sequenced and the amino acid sequences of V3 loop were analyzed by sequence confirm analysis technology. Results The results showed that there were one to ten kinds of polymorphisms in the V3 region of HIV-1 quasi-species which were found from the serial samples of the five LTNP. However, the sequences of the predominant strains were either completely consistent or at most changed at one or two residues in the serial samples of individual patient. The tetramer compositions of the tip of V3 loop were consistent in each patient. It was GPGR in four patients and GPGK in one patient. It was speculated the co-receptor of HIV-1 was CC chemokine receptor (CCR)-5 based on the amino acids at the residue 11 and residue 25 of V3 loop and the net charge of V3 loop. Conclusions There are various polymorphisms at the HIV V3 loop in LTNP. However, the tetramer composition of the tip part of V3 loop is stable. The LTNP are very likely infected with non-syncytium inducing (NSI) strain.
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Objective To analyze the immunogenicity of selected B-cell epitopes of Epstein-Barr virus (EBV) latent membrane protein-2 (LMP2). Methods Three potential dominant B-cell epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 had been predicted using bioinformaties methods. The gene fragments of three epitopes were cloned respectively into pET32a(+) vector and transformed into E. coli strain BL21 (DE3). After identification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the expression products were purified by Ni-NTA agarose affinity chromatography. BALB/c mice in immunized groups were immunized by multi-point intracutaneous injection with the three purified epitope proteins,respectively; and mice in control groups were injected with pET32a (+) protein or phosphate buffered saline(PBS), respectively. The sera from mice at week O, week 3 and week 6 of injection were collected for determination of epitope-specific antibody IgG by enzyme linked immunosorbent assay (ELISA) using epitope proteins as coating antigens. The ability of serum antibody recognizing nature EBV antigen was determined at week 6 of immunization. Results Three epitope proteins of LMP2199-209 ,LMP2318-322 and LMP2381-391 were successfully expressed in prokaryotic system. Epitopespecific antibodies IgG could be detected respectively in the sera of all immunized mice, and the levels of antibodies increased with immunized time increasing. The antibody levels in LMP2318-322 immunized group at week 3 and week 6 were significantly higher than that of pET32a (+) protein control group (F= 493.85 and 773.99, respectively; both P＜0. 05), and the antibody levels in LMP2381-391 immunized group at week 3 and week 6 were also significantly higher than that of pET32a (+) protein control group (F= 926.33 and 309.14, respectively; both P＜0.05). Antibody level in LMP2199-209 immunized group at week 6 was significantly higher than that of pET32a ( + ) protein control group (F=87.27, P＜0.05). The antibody IgG in serum from immunized mice with three epitope proteins could all recognize nature EBV antigens, especially LMP2199-209 and LMP2381-391 immunized groups.Conclusions Three possible dominant epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 are prepared by prokaryotic expression system and exhibit obvious immunogenicity, which could be used for further research of EBV infection and related tumor vaccine.
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Objective To screen proteins from human pancreas cDNA library,which interact with hepatitis C virus(HCV)E1 protein.Methods The human pancreas cDNA library was amplified,purified and evaluated,and then the purified library plasmids were transformed into yeast strain Y187.The reconstructed plasmid pGBKT7-E1 was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp.The transformed AH109 mated with Y187 that contained the library plasmids.The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-α-gal for selecting.The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5α.The plasmids in DH5α were extracted,sequenced and blasted.Result Sixteen proteins interacting with HCV E1 were found.Conclusion Some of the sixteen pancreatic proteins may be related with metabolisms of glucose and lipid.
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Objective To construct and express ribonuclease-resistant virus-like particles containing long chimeric RNA sequence by changing quantity and affinity of ms2 19mer stem-loop.Methods 1.7 kb maturase and coat protein gene of ms2 were amplified.We used a C-variant aptamer of the wild-type stem-loop where uridine-5 has been substituted by cytosine.The 1700 bp PCR-amplified DNA fragments was gel-purified and then digested with BamH Ⅰ and Hind Ⅲ and ligated to linearized pET28b vector to generate recombinant plasmid pET-MC-pac.The five target DNA sequences were spliced by overlapping extension(including 3 fragment of SARS-CoV,one fragment of HCV and one fragment of HSN1).PCR was carried out using primers contained Not Ⅰ site and then PCR were cloned into a pGEM(R) T-easy vector and then excised with the Not Ⅰ restriction enzymes from the resulting recombinant plasmid.simoutaniously the pET-MC-pac plasmid was digested with Not Ⅰ restriction enzymes,fragments were ligated into the linearized pET-MC-pac plasmid to produce a new donor plasmids pET-MC-3V.Simoutaniously,we constructed three recombinant expression vector for control,including N-P3V-pET-P、N-P3V-pET-Cand P-3V-pET-P.N-P3V-pET-P contained one wild type pacsite located behind ms2 sequence:N-P3V-pET-C contained one C-variant pacsite located behind ms2 sequence:P-3V-pET-P contained two wild type pacsites that one is located behind ms2 sequence,another is located between SARS-CoV 3 and HCV sequence.Then these four expression vector were transformed into E.coli strain BL21(DE3),respectively.The 3V Armored RNA was expressed and purified.A260 absorbance value of expression product was determined.Results Four expression plasmids were constructed successfully.The Armored RNA with 1891 bases was snccessfully expressed by the two-paesite expression system of pET-ms2-3V and P-3V-pET-P;for N-P3V-pET-P、N-P3V-pET-C,only 1 200 bp target sequence was packaged into virus-like particles.Expression efficiency of N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P and N-P3V-pET-P was 0.23,0.35,0.35,0.51 mg/ml,respectively.Armored RNA was shown to have the charaeterization of Ribonuclease-resistant and stable at 4℃,37℃ and 25℃ respectively.Conclusion The expression plasmids containing two pacsites were constructed successfully and prokaryotic expression system can be used as an expressing plasmid platform for preparation of Ribonuclease-resistant virus-like particles containing long chimeric RNA sequence.
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Study on protein expression and antigenicity detection of hepatitis C Virus envelope protein E2 in prokaryotic and eucaryotic expression systems.The gene that encoding.HCV E2 protein was cloned in pQE30 and pEF1/HisC.After the expression of E2 protein in E.Coli M15 and COS 7 cell,the expressed proteins were used to detect their antigenicity with ELISA and WB.The results showed that protein E2 was expressed in both prokaryotic and eucaryotic cells.Special reaction could be detected using the expressed proteins and sera from HCV infected people.The studied E2 gene could express the desired proteins in both prokaryotic and eucaryotic expression systems,and glycosylation of the E2 protein happened in COS 7 cell.