2005 Vol.20(3)
To study the immunogenicity of spike protein of SARS coronavirus, an expressional plasmid was constructed encoding partial sequence of SARS Coronavirus spike protein from 2170 bp to 2814bp, named fragment-2(S2). The gene sequence encoding S2 was amplified by RT-PCR from SARS Coronavirus genome RNA, cloned to pMD18-T vector and subcloned to pGEX-4T-2. E.coli JM109 that contained recombinant expression vector pGEX-S2 was amplified and fusion protein expression was induced by 1mmol/L IPTG at 37℃. Protein was extracted and purified by GSH-Sepharose affinity chromatography. After identified by the serum of SARS patient by Western-blot, the purified protein GST-S2 was used to immunize NIH mice three times at two weeks interval, the immunized mice produced high titer anti-GST-S2 polyclonal antibody. It lies a basis for the future study of subunit vaccine of SARS coronavirus.
In this study, α22, an immediate-early gene of HSV-1 was analyzed with RNAi technology. Two sequences of siRNAs (siRNA-1 and siRNA-2) were prepared by in vitro transcription, and transfected into the Hep-2 and KMB17 cells. The cells were subsequently inoculated with HSV-1. The results demonstrated that there was no differences between the transfected Hep-2 and the untransfected cells. Meanwhile, KMB-17, a kind of restricted cell, which was transfected respectively or co-transfected with siRNA-1 and siRNA-2, showed delayed cytopathic effects with the peak titre of offspring virus appearing later. The results suggested the two siRNAs can interfere with the expression of ICP22 and further effect on the replication of HSV-1.
The expression of TGF and its role in the autopsy lung tissue of patients with Severe acute respiratory syndrome (SARS)was investigated .Pathological features of 2 autopsy lung tissues of SARS cases were studied by light microscope. TGF-β-1 expression in autopsy lung tissues from patients of SARS and control lung tissues was analyzed by semiquantitative method using immunohistochemistry staining. In one case, the major pathological changes of autopsy lung tissue were diffuse alveolar damage ,hyaline membrane formed and alveolar exudative inflammation. In the other case, early pulmonary fibrosis and alveolar organization were presented except degeneration and exudation. The mean greym of TGF-β-1 in autopsy lung tissues of SARS was 103.43±0.62, 131.47±2.64 in lung tissue of lobular pneumonia, 144.24±0.09 in normal lung tissue. There were significantly differences among three groups(P0.05). The SARS-coronavirus can lead to acute pulmonary interstitial and alveolar exudative inflammation. In addition, the alveol
A real-time TaqMan-based RT-PCR assay was developed to rapidly detect the Severe acute respiratory syndrome-associate coronavirus(SARS-CoV). Primers and probes specific to the conserved region of the SARS-CoV genome were selected,and the reactive system and conditions were optimized to improve the sensitivity、specificity and repetition of the assay.The results showed that the real-time RT-PCR assay was specific and there were no cross reactions with influenza A-1,A-3,B and H-5N-1 virus、measles virus and other common respiratory viruses.The sensitivity of the assay was 0.1TCID-{50}.It took only three hours from viral RNA extraction to complete the real-time PCR,and the assay was simple and had good repeats. This real-time RT-PCR TaqMan-based assay was a specific、sensitive and quick tool suitable for early and quick detection of SARS-CoV in clinical labs.
A recombinant HCV RNA-dependent RNA polymerase (RdRp, NS5B protein) was expressed in E. coli strain BL21(DE3). The purified protein was used to investigate the RNA syntheses from the 3′ terminal sequences of HCV positive and negative strand RNA in vitro. The positive strand RNA did not direct the synthesis of negative strand RNA; however, the negative-strand RNA was able to generate a full-length positive strand RNA. These results demonstrate that NS5B has the template specificity on negative strand RNA. The template specificity was further confirmed by template competition assay. The competition of positive-strand RNA did not affect the RNA synthesis from the negative strand RNA. The template specificity of NS5B provides a reasonable explanation for the excesses production of positive over negative strand RNA during the HCV genome replication. In addition, these findings provide some clues to further work. The research on viral or cellular factors involved in the RNA synthesis from positive-strand RNA, as we
To understand the differentially expressed target genes in HepG2 cells transfected with PS1TP1 protein expression vector, we compared the differentially expressed genes between the hepatoblastoma cell line HepG2 transfected by pcDNA3.1(-) and pcDNA3.1(-)-PS1TP1, respecti vely with cDNA microarray . The PS1TP1 coding DNA fragment was amplified with polymerase chain reaction (PCR) . The expressive vector of pcDNA3.1(-)-PS1TP1 was constructed by routine molecular biological methods. The HepG2 cells were transfected by pcDNA3.1(-) and pcDNA 3.1(-)-PS1TP1, respectively ,using FuGENE6 Transfection Reagent. Total RNA was isolated and reverse transcribed. The cDNAs were subjected for microarray screening with 4096 cDNA probes. From the scanning results, it was found that 8 genes were up-regulated and 14 genes were down-regulated by PS1TP1 protein expression. The expression of PS1TP1 protein affected the expression spectrum of hepatocyte.
To investigate the effect of SDF-1 gene immunization on immune response induced by HIV-1(Human immunodeficiency virus) nucleic acid vaccine and to explore new strategies for therapeutic HIV DNA vaccine. Balb/c mice were immunized with pCI-neoGAG alone or co-administered with the DNA encoding for SDF-1.Their sera were collected for analyzing anti-HIV antibody and IFN-γ by ELISA , and splenocytes were isolated for detecting antigen-specific lymphoproliferative responses and specific CTL response by MTT assay and LDH assay ,respectively. Our result showed that the anti-HIV antibody titers of mice co-immunized with pCI-neoGAG and the DNA encoding for SDF-1 were lower than that of mice immunized with pCI-neoGAG alone(P0.01).The IFN-γ level of mice co-immunized with pCI-neoGAG and the DNA encoding for SDF-1was higher than that of mice immunized with pCI-neoGAG alone(P0.01).Furthermore, compared with mice injected with pCI-neoGAG alone, the specific CTL cytotoxity activity and antigen-specific lymphoproliferative
Our previous studies showed that the NSP4 gene of human rotavirus epidemic strains in China can be divided into two groups:Wa and Kun, and the Wa group contains three subgroups. To investigate whether there were differences in virulence among the different rotavirus NSP4 genotypes, NSP4 genes from different genetic types were expressed in recombinant baculoviruses using Bac-to-Bac expression system and four recombinant baculoviruses were obtained. Compared with the mock infected cells or the wild type baculovirus AcNPV infected cells, the intracellular Ca 2+concentration increased 5.24, 5.43, 5.34 and 5.45 folds at 72h post infection, respectively. However, there was no significant difference among the increasing of intracellular calcium level induced by the four recombinant baculoviruses. On the basis of the above results, the recombinant NSP4 proteins from Wa or Kun groups were expressed in E.coli and purified ,and the diarrhea induction activity was tested in sucking mice model. The results showed that dia
The envelope glycoprotein E2 of classical swine fever virus(CSFV) was an important protective antigen with more than thirty hydrophobic amino acids at the carboxyl terminal. E2 genes with different lengths of transmembrane region(TMR) of Hog cholera lapinised virus(HCLV) were amplified by RT-PCR,and cloned into pGEX-4T-1 to construct recombinant plasmid pGEXTE2-339, pGEXTE2-355, pGEXTE2-375, respectively. In view of codon usage in prokaryocyte, these plasmids were transfered into E.coli BL21-CodonPlus(DE3)-RP. After induction by IPTG, pGEXTE2-339, pGEXTE2-355 were successfully expressed as inclusion bodies, but pGEXTE2-375 was obviously not expressed. It was showed that TMR could decrease expression ability of HCLV E2 gene in E.coli.Furthermore,after inclusion bodies being washed and denatured, the fusion protein was used as antigen to detect antibody of CSFV and indirect ELISA was developed.
To provide convenient and effective tool for differentiating analysis between EIAV vaccine and pathogenic strain, molecular marker of FLAG+{TM} or 6+*His was inserted into S2 gene of EIAV(equine infectious anemia virus) vaccine infectious clone pFD3, The resulted chimeric clones pFD3-FLAG and pFD3-HISADD were used to transfect fatal donkey dermal (FDD) cells. After 5 generations of in vitro passages, the supernatant was collected to further infect donkey blood leukocyte (DL) for 3 generations of passage. The replicative characteristic of pFD3-HISADD was similar to its parental clone pFD3, RT activity assay was strong positive with significant cytopathtic effect, and the virus particles could be observed under electron microscope. However, pFD3-FLAG showed a lower replication activity, RT activity was weak positive and no significant cytopathtic effect was observed. These resuts implicated that S2 gene was an important factor for viral replication in DL cell.
Two M gene sequences of Canine coronavirus (CCV), from a wild strain NJ17 from a diarrhea dog faecal sample in Nanjing City and CCV 1-71 reference strain, were cloned and sequenced. The results showed, CCV 1-71 shared high similarity (98.9~99.5%) to CCV V1, V2 and a CCV strain from giant panda isolated in China 2003. It suggested that the above CCV strains existed quasispecies from a same ancestor strain. On M gene, CCV NJ17 shared 87.0~91.9% identities with other CCV strains and typical strains of Transmissible gastroenteritis virus (TGEV) of swine, and Feline coronaviruses (FCoVs) in the GenBank. The CCV NJ17 might be a separated evolutionary strain in China. Recombination mark sequences of all the landed strains on the M gene putative recombination region had a “CTTTAG” nucleotides , meeting to “CTT(A/T)(A/T)G” that always appeared near to the recombination “hot spot” in Infectious bronchitis virus RNA genome. The first 50 amino acid residues on N terminal of CCV NJ17 M protein was higher similar to FCoV 7
To study the biological medicine function between BTV and BTV dsRNA molecule of inducing Hela cell to produce IFN and apoptosis in vitro, we carried out the Hela cell monolayer seperately with BTV and BTV dsRNA packed lipofectamine in different concentration level, and detected separately the Hela cell survival rate; the IFN content in Hela cell suspension and the cell apoptosis under the different management by MTT; Elisa Kit and flow cytometor. Analyzed the differences between BTV with different virus dosage and BTV dsRNA-lipofectamine compound with different concentration, we found that the IFN content of Hela cells produced had significant differences under above managements (p0.05), and the ability to induce Hela cell to apoptosis of BTV dsRNA-lipofectamine compound was lower than that of BTV(p0.05). These results manifested that BTV dsRNA could induce Hela cells to produce IFN and apoptosis, and possessed the potential to become a new IFN inducer.
The short interfering RNAs(siRNA) specific for conserved regions of avian Infectious bronchihitis virus(IBV) genome were designed using the software online. The siRNA had the potency of inhibiting IBV production in both cell lines and embryonic chicken eggs. 12 siRNAs from polymerase gene (pol), M and N gene were used for the experiments. N1221 from N gene and Pol50 from pol gene were shown to have an obvious RNAi effect on IBV production in Vero cells and 9 day-old SPF chicken embryos. The RNAi effect depending on the presence of a functional antisense strand of the siRNA duplex showed the positive relation with transfection dosage. It suggested that viral RNA or mRNA was the target of RNAi. This report firstly revealed RNAi phenomenon of IBV replication procedure in Vero cells and 9 day-old SPF chicken embryos.
In this study, sequences of the 1.8kb gene family of 12 different strains of Marek′s disease vivuses were compared. These strains covered 4 different pathotypes, i.e, vaccine, virulent, very virulent, and very virulent plus strains, plus three field strains isolated in China. The results demonstrated that the homology of the upstream regulating sequences of the gene family was more than 92.5% among 12 strains, vaccine strain CVI988/Rispens had more mutations especially with a “CTCGG" deletion between TATA box and a SP1 enhancer. There were 3-7 consecutive repeats of 132 bp in the 1.8 kb gene family of vaccine strain CVI988/Rispens, but only 2 copies were detected in the left strains of all pathotypes. However, there were also only 2 copies of 132 bp repeats found even in Chinese vaccine strain 814, indicating that expansion of 132 bp repeat copies did not have an important role as suggested before. The homology of 1.8 kb gene family was more than 97% among different strains. There were 2 ORF in the 1.69 kb no
One strain of Avian Infectious Bronchitis Virus(IBV) was isolated from layer flock which had the symptom of egg drop in Tai’an of Shandong Province, and identified by electron microscope, HA test and animal regressive research. Results of virus neutralization test showed that the isolated virus is a variant strain of IBV. The whole sequence of S1 gene was cloned and analyzed by RT-PCR using specific primers which can differentiate between 793/B IBV and other serotypes of IBV. The strain′s serotype can be primary deduced as 793/B.
Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus subtype H5 was developed,comparing with virus isolation in embryonated chicken eggs. The NASBA technique described here can detect three isolates of infuenza A subtype H5 and do not detect infuenza A subtype H1,3,6,9, other avian viruses and allantoic fluid of SPF chicken, showing rapidness and high specificity for identificating infuenza A subtype H5 viruses. Dilution of a known virus was used to determine the limit of sensitivity for both NASBA and classic virus isolation. The NASBA/ECL method was equivalent in sensitivity to virus isolation in eggs,with limit of 10+{-1.5} ELD-{50} dose. Virus was isolated from anal swabs, blood and tissues of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). NASBA/ECL could detect viral nucleic acid in anal swabs from the day following artificial infection until death. Conversely, Nucleic acid molecule
The complete HA gene of AIV H9N2 subtype was amplified with PCR method using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank. The PCR product was successively cloned into pMD18-T and pET-32a (+) to get a prokaryotic expression vector pET-HA. The target gene was successfully expressed in the E.coli BL21(DE3) host cell in inclusion body when induced with IPTG. The expression was optimized with proper inducing conditions of 0.7 mmol/L IPTG and 3 hours induction. The highest expression of the target protein was up to 30.8% of the total bacterial proteins. Western-blot analysis proved that the recombinant protein had good immunoreactivity against H9 subtype AIV antibody. The indirect ELISA method for the detection of H9 subtype AIV antibody in chicken serum was established after the optional working circumstance for the ELISA assay (antigen concentration:4μg/mL;serum dilution: 1:80 ) was tried out with chessboard titration . The positive criterion of this ELISA method
Baculoviruses and entomopoxvirus are the viruses which have been reported so far having ubiquitin gene. The ubiquitin gene of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus(HaSNPV) was amplified by PCR. Nucleotide analysis showed that it was 252bp,encoded 83 amino acids with a predicted size of 9.24kDa. The PCR product containing ubiquitin gene was inserted into pET-28a expressive vector. The Ubi-His-tag fusion protein was expressed in E.coli BL21(DE3).The fusion protein was identified by Western blotting with His-tag antibody. The expressions conditions including the contentrations of IPTG and the inducing times were optimized to achieve optional expression. The antibody anti the HaSNPV ubiquitin was produced in rabbit with purified ubiquitin fusion protein , and further study on the function of HaSNPV UBI is underway.
The nonstructural protein gene 3ABC of Foot-and-mouth disease virus (FMDV) was amplified by reverse transcription (RT) PCR and the PCR product was inserted into transfer vector pFastbacHT. Then, the recombinant vector pFastbac HT-3ABC was extracted and transfered into DH-{10}Bac containing a shuttle vector Bacmid. 3ABC gene was integrated into Bacmid by site specific transposition. Subsequently, recombinant shuttle vector Bacmid-3ABC was transfected with Hi Five insect cells. Identification by PCR amplification demonstrated that 3ABC gene was correctly inserted into baculovirus genome at the downstream of polyhedrin promoter. SDS-PAGE and Western blot analysis detected a band of about 50 kDa in the expression product of 3ABC gene in insect cells. The successful expression of 3ABC gene in Bac-to-Bac expression system should provide a new technical platform for the development of novel approaches for distinguishing the infected animals from vaccinated animals.
The coat protein gene of Cucumber mosaic virus (CMV) from melon isolate (CH99/ 90) was amplified by RT2PCR from the total RNA of infected zucchini leaves and cloned into pUCm2 T vector . The gene consisted of 657 nucleotides encoding 218 putative amino acids. Nucleotide sequence alignments showed that the CP gene shared 92. 2 %～ 93. 9 % homology wit h that of CMV subgroup I strains , whereas only 76. 8 %～77. 8 % homology with that of CMV subgroup II strains. The nucleotide sequence shared 91. 8 %～93. 4 % homology with CMV isolate previously reported from China except for XB isolate. This melon isolate was classified into subgroup I based on these data. The gene was constructed into plant expression vector by using intermediate vector pJIT163 ( recombinant plasmid designated as pBCP) . The recombinant plasmid was transferred into competent cells of Agrobacterium tume faciens LBA4404. Transformation of the gene into watermelon is undertaking.
The DNA component 5 (DNA-5) of Banana bunchy top virus NS strain was cloned and sequenced. It was 1014nts in full length and could code for 146 amino acids. The nucleotide and amino acid sequences of the DNA-5 were compared with those of isolates from Australia, Hawaii, Egypt, and identities of nucleotide and amino acid sequence among these isolates were between 88%~89% and 80%~88%respectively. The secondary structure near the “LXCDE” motif was much different between NS strain and others. It was deduced that efficiency of binding Rb protein would be influenced by this kind of difference.
The nucleotide sequences of full length RNAs of CMV-CA strain were determined and compared with those of CMV-Fny in subgroupⅠA, CMV-SD in subgroupⅠB and CMV-Q in subgroupⅡ, and Peanut stunt virus(PSV) ER strain in the same genus. CMV-CA RNA1 is 3356nt long,containing 1 ORF encoding 111kDa 1a protein; RNA2 is 3045nt,containing 2 ORFs encoding 96.7kDa 2a and 13.1kDa 2b proteins, RNA3 is 2 219nt,containing 2 ORFs encoding {30.5}kDa 3a and 24kDa CP. The sequence similarity of CMV-CA RNAs with the corresponding RNAs of CMV SD,Fny,Q strains were as following: RNA1∶91.3%,91.1%,76.5%; RNA2∶92∶1%,90%,71.2%; RNA3∶96.1%,92.6%,74.5%,respectively. The sequence similarity of CMV-CA RNAs with the corresponding RNAs of PSV-ER were 67.1%,58.2% and 55.7% respectively. These results showed that CMV-CA was not the reassortment virus of CMV with PSV, although both of two occur in peanut fields in northern China. Based on RNA3 5′NTR structure, and phylogenetic tree analyses of RNA3 5’NTR nucleotide sequence and CP amino acid sequence , CMV2CA is classified in CMV IB subgroup
The HC-pro gene was amplified by RT-PCR from total RNA of tobacco leaves infected with a N strain of Potato virus Y in Shaanxi, and cloned into the PMD 18-T vector. This HC-pro gene is consisted of 1371 nucleotides, encoding 457 amino acids. It shared the sequence homology of 82.5%~96.4 % nucleotide acid and 92.5%~98.0% in amino acids compared to 9 species of PVY N HC-pro abroad.The HC-pro gene was inserted into prokaryotic expressing vector pBV221, to obtain pBVHC recombinant plasmid in E.coli BL21. SDS-PAGE indicated that HC-pro proteins are successfully expressed in E.coli, Western blotting analysis demonstrated that the antibody against the expressed HC-pro can be used to identify the infected plants .
The gene of nucleocapsid protein of Equine arteritis virus was amplified from PMD-18-T plasmid with equine arteritis virus ORF7 sequence by PCR. The PCR product was sequenced as well as purified and digested with EcoR I and Xho I, then directly cloned into the prokaryotic vector pET32a. Consequently the recombinant plasmid was constructed, designateds pET32a-N. PET32a-N was transformed into the host cell BL21(DE3) and the expression procedure was optimized including cultivated temperature, optional induction concentration and time of IPTG. The result indicated that the nucleocapsid protein can be expressed efficiently with 0.8mmol/L IPTG and 4 hour induction. The resulting Trx-N recombinant fusion protein was identified to be consisted of 34 kDa protein by SDS-PAGE and western blotting analysis. It indicated that the recombinant fusion protein could be used as antigen of diagnostic assay for detecting antibodies.
Influenza A virus is a widespread pathogen,but current vaccines and drug therapy are of limited value. RNAi may provid a novel route to control it since RNAi can inhibit gene expression high-efficiently and specificly. A recombinant plasmid with a short hairpin RNA(shRNA)targeting at the nuclear protein gene of the virus was constructed and identified by enzyme digestion and sequence analysis.The hemagglutination(HA) titre and TCID 50 of the virus in chicken embryo fibroblastic(CEF)cells transfected with the plasmid was lower than that nontransfected with the plasmid.It showed that the plasmid could specifictly inhibit the virus production in CEF cells.
In the course of calculation of genetic distance of virus gene, p and q should be comprehended of the transition rate and transversion rate. The result K should response the time concept of evolution speed if K would be a percent. So genetic distance were proposed to change into evolution per cent, then exploring expediently the trace of virus evolution from the disciplinary change of evolution rate.
