We analyzed and verificated the B-cell epitope of the L1 capsid protein of the Human papilloma virus type 6, and made it the base of producing new subunit peptide vaccine. Goldkey and PC/ Gene computer program were used to colligate and analyze the B-cell epitope of HPV6 L1. These peptides were synthesised and purified by Fmoc and HPLC methods, respectively. After that the peptides were emulsified with 0.2ml adjuvant, the blood serum antibody titer of mouse immuned by 50 μg peptides were detected. We found that, the B cell epitopes of the L1 protein of HPV-6 could be located at NO.425-439 and 486-500 positions. Those synthesized peptides have the ability to raise blood serum antibody titer enormously in vitro, and the antibody induced by these synthesized peptides could react with the supernatant of human papilloma tissue. We conclude that NO. 425-439 and 486-500 positions are predominat B-cell epitope of HPV 6 L1 protein. More work is under way to investigate whether these antibodies could perform functions.
To create the pathogenicity model of severe acute respiratory syndrome (SARS) on macaca rhesus, the macacu rhesus was infected with SARS coronavirus (CoV) was detected in different samples coming from macaca rhesue in different days by virus isolation, immunoflurescence assay, pathological inspection and reverse transcription polymerase chain reaction (RT-PCR). The results showed that SARS CoV were isolated from the above samples, and SARS CoV RNA could be detected in blood of the 2nd and 5th day, secreta of nose-throat of the 7th and 9th day, faeces of the 3th day, and faeces and urines of the 5th day after infection. Under the microscope, it was found in the group infected by SARS CoV that pulmonary alveolus interval had been broadened and had many lymphocytes and monocytes infiltrated. There was much exudation in the chamber pulmonary alveolus, which even formed the hyaline membrane. The organized pneumonia could be found in several pulmonary alveolus. great necrosis foci was also observed in the liver, companied by some inflammatory cells infiltrated. It may be successful to create the model of SARS on macaca rhesus, and show that the animal model can be used to evaluate anti-SARS CoV drugs and vaccines.
To understand the relationship between the chronic hepatitis B virus infection and the primary hepatocellular carcinoma (PHC), we adopted the method of retrospection and carried out analysis by comparing the markers of Hepatitis B virus (HBV M) and the result of liver function testing of the 328 cases of PHC and 340 patients with the enteron tumor not suffered from PHC, all of whom were treated in Wuhan general hospital of Guangzhou command of PLA from Jan, 2000 to July, 2003. The results showed that the positive rate of HBsAg in patients with PHC (63.11%) was significantly higher than that in control group with non-PHC (other enteron tumors) (11.47%). In the cases of PHC, the positive rate of the combination of HBsAg, HBeAb and HBcAb was presented 37.2%, which was significantly higher than that of the combination of HBsAg, HBeAg and HBcAb. According to the results of liver function, there was no significant difference between the group which were the combination of HBsAg(+), HBeAb(+), HBcAb(+) and the group which were the combination of HBsAg(+), HBeAg(+) and HBcAb(+) (P0.05). Hepatic injury in cases of PHC was significantly higher than that in control group of non-PHC (P0.01). So the chronic hepatitis B virus infection plays extremely important role in PHC’s etiology. The human group who were positive with HBsAg, HBeAb and HBcAb is the PHC’s high-risk group.
Although severe diseases may be caused by respiratory syncytial virus (RSV) infection in the world, no efficacious vaccine against this virus is licensed. In an effort to seek recombinant protein antigens that may be used in the future vaccine development, we constructed a series of expression vectors which can co-express carrier proteins and G protein fragments based on cloning the full cDNA sequence of G protein from RSV. An expression system that can efficiently express recombinant protein antigens in soluble form was selected for subsequent researches. Balb/c mice were immunized with one of the purified recombinant protein antigens, DsbA-G101, for production of anti- serum. Based on ELISA assays, good immunogenicity of DsbA-G101 was revealed. The constructed expression vectors can be used in immunogenicity analysis of different G protein fragments and in selection of ideal carrier proteins, two aspects which are important for the future development of vaccine against this virus.
The recombinant expression plasmid of pVAX1gag-gp105 was constructed and transfected into BHK21 cells by lipofectamine, the expressed product was detected by indirect immunofluore scence. After the mice were immunized with pVAX1gag-gp105 and control plasmid, the number of CD4＋and CD8＋ subgroups of spleen T lymphocyte, the specific killing activities of spleen CTL and the titers of serum antibodies of the immunized mice were detected. Compared with vaccinated control groups, pVAX1gag-gp105 vaccinated group had significant differences in the titers of the serum antibodies (P0.01), the numbers of spleen CD4+ and CD8+ T cells (P0.05) and the specific killing activities of spleen CTL (P0.01). These results manifested that the DNA vaccine based on HIV-2 chimeric gene gag-gp105 possessed good humoral and cellular immunogenecity in BALB/c mice.
The recombinant retroviral vector pBABE-puro-E1E2 was constructed by inserting the full-length HCV e1e2 gene of H77 strain into pBABE-puro. Both the recombinant retroviral vector and the pVSVg plasmid were transfected into eukaryotic cells 293T by calcium phosphate transfection method. And then, the pseudovirus were produced. The pseudovirus infected eukaryotic cells SP2/0 and E1E2 protein was expressed. E1E2 protein was detected by puromycin-resistant and FACS analysis. BALB/c mice were injected in abdomen with expressing E1E2 protein SP2/0 cells. Anti-HCV E1E2 antibody was screened by FACS. Moreover, the antibody was also analyzed by Western blot using E2 protein antigen which was expressed in E. coli. The results showed that HCV E1E2 protein was expressed in SP2/0 cells’ envelope successfully. FACS could detect specific anti-E1E2 antibody in SP2/0 cells immune mouse serum. Western blot analysis showed that SP2/0 cells immune mouse serum could react specially to E2 protein that was expressed in E.coli.
vp2 gene of Porcine parvovirus (PPV) SD-68 strain was cloned and sequenced .The results showed that vp2 gene of Porcine parvovirus (PPV) SD-68 strain was 1740bp and encoded a protein of 579 amino acids. vp2 gene of SD-68 strain exhibited over 99% nucleotide sequence identity and 96% amino acid sequence identity with other PPV strains: Kresse, SY-99, NADL-2(5075), NADL-2(4973), US-1. Phylogenetic tree analysis of vp2 gene showed the closest relationship between SD-68 strain and kresse strain. The difference between SD-68 strain and kresse strain was only on 378, 383 and 436 residues, which decided the tissue tropism of PPV, so the tissue tropism of SD-68 strain suggested more similar with kresse stain, that is to say, SD-68 was a dermatitis strain. Comparison of amino acid residues of virulent strain (kreese) and vaccine strain (NADL-2 and SD-68) suggested that 215, 378 and 383 were important residues that decided the pathogenicity of PPV.
White spot syndrome virus ORF220 encodes a protein homologous to the gp130 receptors of eukaryote. prcvious studies showed that this protein was a minor structural protein. In this paper, the ORF220 was cloned into the pFastBacI fused with EGFP gene and co-transfected with AcBacmid into DH10B cells. The recombinant Bacmid containing fused ORF220 and EGFP genes was confirmed by PCR and then transfected into Sf21 cells. The green fluorencence was observed 3-5 d post-transfection under the fluorescent microscopy and this confirmed that the fusion protein was expressed in insect cells. The supernatant of the transfected cells was used to infect the Sf21 cells and the time course observation showed that the ORF220 product localized in both the cytoplasm and nucleus with no predilection.
In order to define if similar cytokines take part in transactivation of Jembrana disease virus and Human immunodeficiency virus-1 Tat protein, two truncated Tat proteins, hTat47 and jTat70, which contained intact activation domains but lacking binding domains, were constructed. We also constructed cyclin T1 and CDK9 eukaryotic expression and antisense plasmids. Overexpression of hTat47 and jTat- 70 both inhibited transactivation of JDV and HIV-1 Tat on HIV-1 or JDV LTR, suggesting that they may involve similar cytokines. Antisense transient assay of cyclin T1 and CDK9 further proved that these cytokines also participated in transactivation of JDV Tat.
Here we report that the major antigenic protein VP2 of very virulent Infectious bursal disease virus (vvIBDV) was secretively expressed with high level in yeast (Pichia pastoris) expression system. Primers were designed to amplify the previously cloned vvIBDV-vp2 gene from the plasmid pMD18-T- VP2. The amplified 1.4kb vp2 gene fragment was sub-cloned into the yeast-expressing plasmid pPICZα-A. The recombinant pPICZα-A-VP2 was analyzed by restriction enzymes and sequenced, which was then linearized by SacⅠand transfected into Pichia pastoris X-33. After selection with ZeocinTM, the phenotype identity, the inductive expression, the SDS-PAGE and the western blot analysis of culture supernatant, an engineering Pichia pastoris strain X-33/pPICZαA-VP2 with which the VP2 protein could be expressed with high level was obtained. SDS-PAGE and Western blot analysis indicated that the expressed product of the VP2 in culture supernatant of X-33/pPICZαA-VP2 was about 55kDa, a bit larger than expected. Gel scanning and Bradford protein analysis showed that the expressed product was as high as 23mg/ L, or 28% of total proteins in culture supernatant of X-33/ pPICZαA-VP2.
Two pairs of primers were designed according to the sequences of envelope genes, vp19 and vp28 of White spot syndrome virus (WSSV) in the GenBank. The two DNA fragments about 370bp and 630bp amplified by PCR were linked to the EcoR I restriction endonuclease site and cloned into E.coli expression vector pET- 22b(+) in the proper Open Reading Frame(ORF). With IPTG induction at 35℃, the molecular weight of the engineered protein was about 41kDa, which was identified by SDS-PAGE analysis. Antiserum of the fusion envelope protein VP (19+28) purified by Ni2+- column chromatography was prepared by immunizing rabbit. The VP (19+28) antiserum was used to neutralize the WSSV infection on crayfish by intramuscular injection. The result showed that the antiserum was effective in the neutralization of WSSV on the crayfish which were reared with artificial food at 15-22℃.
Structural and envelope glycoprotein E2 (gp55) of Classical swine fever virus (CSFV) is the most antigenic protein being responsible for eliciting neutralizing antibodies and conferring protective immunity. Infection of cells with CSFV is mediated by the interaction of E2 and Erns with the cell surface receptor. In this paper we report the analysis of E2 epitope by screening a 12-mer random peptide phage display library using the monoclonal antibodies (MAbs), c2410 and a18, raised against CSFV strain alfort Tübingen and reacted with the E2 structural protein. MAbs, c2410 and a18 recognized the same linear epitope, located at aa832- aa 837 (SPTTLR) of E2 protein, but showed some different reactivities with the mimotopes by ELISA and Western blot analysis. Sequencing both cDNA of light chain and heavy chain variable regions of the two MAbs from total RNA of the hybridoma cells was carried out. The results showed that MAb c2410 and MAb a18 are different monoclonal antibodies, though both MAbs derived from the same fusion and recognized the same epitope.
Based on the fact that the envelope glycoprotein E2 which can protect swine from virulent attack of Classical swine fever virus(CSFV) has two structural antigenic domains B/C and A/D, a pair of specific primers was designed to amplify the gene fragment encoding A/D antigenic domain of E2 protein. The 373 bp PCR product was directionally cloned into Pichia pastoris secretory expression vector pPICZαC under the control of the AOX1 promoter and α-factor secretion signal sequence. After being linearized with restriction endonuclease Dra I, the recombinant plasmid was transformed into Pichia pastoris by electroporation. Five transformants with high copies were acquired when selected under ZeocinTM and were induced with methanol. SDS-PAGE indicated that the supernatant of the induced P. pastoris culture contained the recombinant protein E2 (175.8ug/mL). Western-blot analysis proved that the recombinant protein had good reactimmunity against positive CSFV serum. N-glycosylation analysis of expressed products showed that the recombinant protein was glycosylated in the process of secretion. Our research provided a basis to develop sub-unit vaccine and diagnostic antigen against CSFV.
Thymidylate Synthase (TS) is an enzyme involved in making one of the major building blocks for DNA deoxythymidylic acid (dTMP), and plays a critical role in cell growth and division and is overexpressed in the majority of cancers. Lymphocystis disease virus (LCDV) belong to iridoviridae, and the causative agent of lymphocystis disease, which has been reported to occur in over 100 different fish species worldwide, and viral infection that causes cells to become megaloblastic, thus forming small tumors (bumps or growths). The complete nucleotide sequence of the LCDV-C genome was recently determined. Among the sequenced vertebrate iridoviruses, lymphocystis disease virus isolated from China (LCDV-C) is of the largest genome. Computer-assisted analysis revealed the potential open reading frames ORF 011L of LCDV-C was TS gene, the first report of complete TS gene from vertebrate iridoviruses. The LCDV-C TS gene consists of 858 bp in length with a base composition of 28.2% G+C, and encoded a protein of 286 amino acids with a predicted size of 32.7KD. Comparison of the LCDV-C TS to TS from Chilo iridescent virus(CIV), Ambystoma tigrinum virus (ATV) and tiger frog virus (TFV) three iridoviruses and other sixteen species, showed extensive amino acid similarities of TS between four iridoviruses and other species. The predicted LCDV-C TS amino acid sequences contain the folate-binding site in 44-70 residues and dUMP-binding site in 163-206 residues. The secondary structure of LCDV-C TS was predicted with 8 alpha helixes, 6 beta sheets and 23 loops, showed the structure flexible and torsional rotation. Phylogenetic tree was constructed based on the multiple alignments of TS amino acid sequences from twenty species, and LCDV-C TS is a branch alone.
On the basis of construction the transfer vector pgEI-GFP of Pseudorabies virus SH strain BHK-21, which was infected with PRV-SH for 1-2h, were tansfected with the complex of pgEI-GFP and DOTAPA deletion mutant was selected and purified 3-4 times in BHK-21 cell through GFP. In the research here, we investigated the gE-/gI-/GFP+ PRV vaccine strain growth properties in cultured cells, its safety for rabbits, its LD50 for mice, its safety and immunity for postweaning piglets, and its biological properties. The results suggested that gE and gI genes deletion may not affect PRV’s propagation in cultured cells, nor the typical PRV plaque forming. The investigated results demonstrated that the virulence of gE-/gI-/GFP+ was reduced when compared with that of PRV-SH. The inoculation of gE-/gI-/GFP+ couldn’t harm the post-weaning pigs, couldn’t induce the viral spread in surroundings. The period of fever and the number of days of existing virus in treated group pigs were less than that in the control group after big dose of RPV-SH were used to equally attack the treated and control group pigs through ear veins 5 weeks later. gE-/gI-/GFP+could always induce the high titer PRV neutralizing antibodies throughout the experiment.
In order to effectively differentiate between gene-deleted vaccine and wild-type isolates of Pseudorabies virus(PrV), a PCR method, based on sequences of gE and gI of the PrV genome, was established after selection of the optimal reaction conditions. By applying this technique, the 848bp gene fragment was amplified from PrV RA strain, SH strain and LA strain, respectively. The negative results were achieved from Bartha-K61, RK-13, VSV, HCV, PRRSV, JEV, PPV and PCV2. Sequencing of the amplified products showed that the PCR method was specific. The sensitivity of PCR reached to 5pg DNA of PrV RA strain . We applied the PCR method to detect 172 tissue samples from 37 pig farms in Jiangsu, An’hui, Zhejiang, Fujian and Shanghai during 2003-2004, wild-type isolates of PrV were found in 35 tissue samples (20.34%) and 15 pig farms(40.54%). PrV distributed widely in naturally infected pig’s tissues including brain, liver, spleen, kidney, lung and lymphnodes. The PrV positive detection rate in lymphnodes was the highest in all tissues examined and reached 83.33%. The established PCR method provided a more sensitive, specific and reliable method to rapidly detect wild-type PrV and epizootic study of PrV.
In this study, the major antigenic region of spike (S) gene of Canine coronavirus giant panda’s isolate (CCV strain DXMV) was expressed in Pichia pastoris. The S1 gene fragment encoding major antigenic region of S protein was amplified and cloned into T vector, then subcloned into pPICZαA for yeast expression. The recombinant pPICZαAS1 was linearized with SacI and then transformed into competence GS115 cells for expression under the induction of 1% methanol. The positive yeasts were screened by PCR. Expression of S1 protein was identified by SDS-PAGE and Western blot. The results revealed that it had a molecular weight of 106 kDa, which could be specifically recognized by multiclonal antibody against CCV. The expression of recombinant S1 protein amounted to 6.6-8.6% of the total protein of supernatant by gel scanning. After three immunization by the recombinant S1 protein, the neutralizing antibody in sera of mice can range from 1:8 to 1:16, which indicated that the recom- binant S1 protein was of it’s immunogenicity.
Hypervariable region of vp2 gene of six different IBDV (Infectious bursal disease virus) isolates, which were isolated from Jiangsu province, was amplified by RT-PCR and sequenced. Furthermore, sequences of six IBDV isolates were analyzed by ClustalX and Phylip3.5. It was suggested that three isolates (Y3, P2G, P8G) were closely related to vvIBDV (very virulent IBDV) strains isolated from China and Europe. Other isolates (SZ, Y5, W04) were very close with Japanese vvIBDV strains. This study laid on foundation for the research of molecular epidemiology and the development of IBDV vaccines.
One anti-idiotypic monoclonal antibody（a-Id Mab）was raised directed to both rabbit and chicken IgG against Avian influenza virus(AIV). The Mab was able to inhibit the binding of AIV to anti-AIV IgG and specific in binding anti-H9-subtype-AIV IgG. Another experiment demonstrated that the Mab might induce chicken to generate hemagglutination inhibition antibodies. These results indicated this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on AIV.
The nucleotide sequence of Dendrolimus punctatus Wenshanensis cypovirus genomic segment 8 (S8) has been determined. S8 consists 1332 nucleotides and encodes a putative protein of 390 amino acids with a molecular mass of approximately 44kDa (P44). Then, the fragment containing p44 gene was inserted into pET-28a expression vector and expressed in E.coli BL21. SDS-PAGE results showed that p44 gene has expressed.
To investigate the role of actin in AcMNPV BV transportation from nuclear to cytoplasm, we constructed two recombinant viruses AcMNPV-GFP and AcMNPV-GFP-actin, in which the expression of GFP gene and GFP-actin fusion gene was driven by polyhedrin promoter. Here, we report the expression pattern of GFP in Sf9 cells infected with AcMNPV-GFP and AcMNPV-GFP-actin. Within 24-72 hr after infection, we observed that GFP signal in Sf9 cells infected with AcMNPV-GFP-actin was first concentrated in nuclear and then gradually located to cytoplasm and finally accumulated exclusively at cytoplasmic membrane. In the Sf9 cells infected with AcMNPV-GFP, however, the GFP signal was diffused throughout cell and this expression pattern did not change during the same period of observation. Thus, our results indicated that actin might participate in the transportation of the AcMNPV BV from nuclear to cytoplasm and its exocytosis from the cell.
Using a pair of specific primers designed according to the relevant nucleotide sequences from GenBank, the main antigen domain for VP2 gene of Porcine parvovirus was ampilified with PCR method using the genomic DNA as template. The PCR product was cloned into the expression vector pIREShyg to get a recombinant eukaryotic expression plasmid pIREShyg-VP2, which was then transfected into the CHO-K1 cells. The expressed product was detected by IFA after the positive cell clone was selected with hygromycin. The result revealed that the main antigen domain for VP2 gene of porcine parvovirus was stably expressed in CHO-K1 cells.
In this essay the destructive rates of HBsAg, the surface antigen of HBV, exposed to different light sources and photocatalysed time on both TiO2 ceramic plate and Ni net were investigated, the effect of anti-HBV of TiO2 was discussed under different light condition.The result demonstrated that ceramic TiO2 plate has 87.50% destructive rate under high Hg lamp irradiation(8μW/cm2) after 4 hours, while the controlled normal ceramic almost had no influence to HBsAg. Also the result indicated that under indirect sunlight(20μW/cm2) for 4 hours, plate with TiO2 destroyed 93.75% of the HBsAg but the ordinary plate destroyed 87.50%.The Ni net with TiO2, when placed under ultraviolet irradiation (480μW/cm2)for 2,5,10 minutes destructive rates to HBsAg were 99.80%, 99.90%, and 100% respectively, but the controlled net without TiO2 thin film showed only 93.75% even after 10 minutes under the same ultraviolet.