1998 Vol.13(4)
The baculovirus insect cell expression system was used to prepare the HPV16 E 6 recombinant protein. The HPV16 E 6 complete ORF amplified by PCR from HPV16 genome was T A cloned into the baculovirus transfer vector pVL1393 T under AcMNPV Polh. This recombinant transfer plasmid pVL1393 E 6 DNA and baculovirus DNA were co transfected into insect cell Sf 9. After the plaque screening, the recombinant baculovirus AcMNPV E 6 was expressed in insect cells. The E 6, analysised by SDS PAGE, was 18kD, and was recognized by antibody against E 6 protein, which showed their natural antigenecity.
The effect of Polygonum cuspidatum water extract (PCWE) on C57BL/6 mice infected with LP BM5 murine leukemia virus (MuLV) was investigated taking spleen index, viral antigen positive cell, serum IgG level and ConA response as parameters. It is demonstrated that PCWE can partly inhibit LP BM5 MuLV induecd splenomegaly, immunodeficiency and viremia of C57BL/6 mice.Antiviral effect of PCWE is more effective before or at the same time of virus inoculation than after virus inoculation. It indicates that the earlier treatment, the better. Continual treatment can partly improve splenomegaly and ConA response of infected mice, but the inhibition of PCWE on hypergrmmaglobulinemia of infected mice is non persistent.
In order to find new anti human immunodeficiency virus (HIV) ribosome inactivating proteins (RIPs), the anti HIV activities of 47 RIPs and crude proteins from 17 species of plants were examined by the following measurements: 1) the inhibition of syncytia formation; 2) the protection of HIV 1 infected cell;3) inhibition of HIV 1 infected cell fusion in coculture;4) reduction of p24 antigen expression and numbers of HIV antigen positive cells in acutely and chronically infected culture. Among 47 proteins, trichosanthin (TCS) (SI=193.3) from Trichosanthes kirilowii, protein fractions Ⅴ (SI=243) and Ⅵ (SI>1200) from Trichosanthes damiaoshanesis were found to obviously inhibit syncytia formation induced by HIV 1. Crotin Ⅰ from Croton tiglium, luffin from Luffa cylinarica , RIPs from odgsonia macrocarpa and Entada phaseoloides showed slightly inhibition of the syncytia formation (SI<50). Crude protein extracts from Trichosanthes Ovigera, Momordica macrophylla and an unnamed plant growing in
HPV 16 (HB strain) E 7 gene was cloned into expression vector pWR590 1 by gene cloning technique. Recombinant plasmid pWHB E 7 was obstained by analysis of restrictional endonuclease. 70 kD HPV 16 (HB strain) E 7 fusion protein was expressed by E.coli JM 109 transfected with pWHB E 7. The recombinant protein could be recognized by standard E 7 monocloned antibody in Western blot. The production of E 7 recombinant protein was more than 30 percent of total E.coli protein by induction of IPTG. The E 7 fusion protein existing in inclusion body in E. coli could be conveniently extracted and purified.
With in situ hybridization technique the localization and distribution of HSP70 mRNA and Hantaan virus RNA in the tissues of 30 cadavers with epidemic hemorrhagic fever (EHF), which were collected from different EHF epidemic foci in China, were examined. HSP70 mRNA was detected in most of the 30 cases and consistent with the localization and distribution of viral RNA.The distribution of HSP70 mRNA was partly related with the pathogenesis of HFRS. The over expression of HSP70 was also found in the virus infected Vero E 6 cells. It suggested that HSP was associated with the infection of Hantaan virus.
The gene encoding the N terminal 1-65 amino acids of preS1 was amplified by PCR and fused to the 3′ end of hTNF α gene to form a hTNF α preS1 fused gene which was directly inserted into the expression vector pSB 92 between EcoRⅠ and BamHⅠ sites and under the control of P 1 promoter. The fused hTNF α preS1(1-65) gene can be expressed in E.coli by temperature induction. The expression level was up to more than 35% and expressed fussion protein was shown by a major band with a expected molecular weight about 25kD on SDS PAGE, and also confirmed by Western blot analysis with anti hTNFα monoclonal antibody and anti preS1 monoclonal antibody. After renaturation, the fused protien displayed the hTNFα biological activity. The preS1(1-65) gene is found to be accurately fused to hTNFα by sequence analysis.
In order to get high level expression of HIV 1 gp41 gene, a fragment (690 bp) encoding N terminal of gp41 from all sequence of HIV 1 was amplified by PCR. After digesting by restriction enzyme EcoRI/Sal I, the fragment was cloned into pET28a plasmid which was digested by the same restriction enzyme, then recombinant plasmid was transformed into the E.coli BL21 (DE3). After inducing by IPTG, the bacteria produced the protein. Then the protein was purified through chelating Sepharose. Using indirect ELISA, SDS PAGE and Western blot test, it was found that the protein had good antigenicity, good specificity and high purification (99%), and its yield was about 45% of the total bacteria protein.
The multiple with other biological agent Autographa californica nucleopolyhedrovirus (AcMNPV) pesticide, which is widely used to control Spodoptera exigua and Prodenia litura as well as Artogeia rapat, Hellula untalis, Plutella xylostella , was selected. Tested with 2nd instar S. exigua larvae, the virulence of AcMNPV A strain amplified in S.exigua larvae is higher than that of AcMNPV B strain in Sf9 cells. LD 50 of AcMNPV B (1949.4 PIB/Larva) is 14.79 times more than AcMNPV A (131.8PIB/Larva).The ratio of LD 50 between AcMNPV A (LD 50 =168.84-240.36 PIB/Larva) and AcMNPV A+Bt (LD 50 =1.4329-128.91 PIB/Larva) is 118-1.5 while the ratio between AcMNPV A and AcMNPV A+Bt+PlNPV (LD 50 = 21.82 -21.4 PIB/Larva) is 7.7-9.5.
Tested with 2nd instar P.litura larvae, the ratio of LD 50 between PlNPV (LD 50 =16.02-53.53 PIB/Larva)and PlNP V+Bt(LD 50 =2.9-7.76 PIB/Larva ) is 5.5-6.9 while the ratio between PlNPV and PlNPV+Bt+AcMNPV A (LD 50 =3.16-15.469
Screening inhibitors of GCHV 873 (Grass Carp Hemorrhage Virus)to inhibit its replication in CIK cells in vitro ,the biotinylated intact virion of GCHV was used to react on a random sequenced peptide library of 9 amino acids fused on protein Ⅲ of filamentous phage fUSE5 .After 3 rounds of affinity selection,immuno screening and ELISA,16 positive clones were selected using CsCl gradient purified virus particles, 6 of them inhibited the replication of GCHV in CIK cells in vitro and decreased the TCID 50 of GCHV from 10 -10 to 10 -5 .These findings open possibility of identifying virus proteins or epitopes as targets for antiviral agents.
Through the reaction between GCHV 873 (Grass Carp Hemorrhage Virus) particles and random peptide library displayed on phage in vitro, after 3 rounds of affinity selection 16 phage clones from 300 clones have been got, which have high affinity with GCHV. Six of sixteen clones could inhibit the replication of GCHV in CIK in vitro and decrease the TCID 50 to half of index. The DNA sequence of phage clones and deduced sequence of amino acid of the inserted foreign fragments were analysed. The results show that the sequences of six clones are identical. The deduced sequence of amino acid is NH2 Leu Trp Val Gly Gly Gly Arg Asn Ala. It indicates that the ability of anti GCHV is certainly related to specific structural information and amino acid sequence of peptides. The determination of antiviral peptide offers a fundamental practical advance in the synthesis of the antiviral peptides and provides a way of generating noval antiviral agents.
After infecting with Vaccinia virus, HeLa cells manifest typical morphological characters of apoptosis, and the electrophoretogram of their chromosomal DNA shows the ladder pattern. In situ apoptosis detection reveals that the location of the DNA cleavage is near the nuclear membrane, where chromatin condensation also occurs.
A method of antibody capture polymerase chain reaction (AC PCR) is developed for the detection of human hepatitis A virus (HAV) in shellfish ( Scapharca subcrenata, Buditapes philippinarum, Macoma incongrua, Sinonovacula constricta, Chlamys farrer ). Initial virus extraction is based on a modified elution precipitation technique. Eleven out of 31 shellfish samples from Lianyungang (in Jiangsu Province) sea water are HAV positive. This experiment proves that the consumption of shellfish growing in contamination waters is associated with the risk of transmission of HAV.
