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1998 Vol.13(4)



Advances of research on molecular biology of rhabdoviridae II. viral structural protein, transcrip- tion, replication and eapsidation

SHEN Jia-Li, GONG Zu-Xun

1998, 13(4): 281

Advances of Research on Molecular Biology of Barley Yellow Dwarf Virus

HAN Zhi-Yong, ZHAO Zhang-Xing, ZHU Mu-Yuan

1998, 13(4): 292

Human papillomavirus type 16 E6 gene's T-A cloning and expression in insect cell

LUAN Yi, YU Xiu-Ping, ZHAO Wei-Meng, GU Ji-Hui, ZHOU Ya-Bin

1998, 13(4): 305

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.

Evaluation of antiviral effect of Polygonum cuspidatum water extract with a model of murine ac- quired immunodeficiency syndrome

JIANG Yan, WANG Hong-Xia, BAO Zuo-Yi, ZHU Guan-Fu

1998, 13(4): 311

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.

Anti-human immunodeficency virus activities of proteins from 17 species of plants

ZHENG Yong-Tang, BEN Kun-Long, JIN Shan-Wei

1998, 13(4): 320

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

Cloning and expression of E7 gene of human papillomavirus type 16 (HB strain

WU Xin-Xing, ZHAO Wen-Xian

1998, 13(4): 326

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.

Localization and distribution of HSP70 mRNA and Hantann virus in autopsy tissue of the patients with EHF and in virus-infected cells

XIE Lin, YANG Shou-Jing, LIU Yan-Fang, YAN Pei-Song

1998, 13(4): 332

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.

Fused expression of hepatitis B virus preS1 gene in Escherichia coli

CHEN Bo, LIU Hui, REN Gong-Yu, SU Cheng-Zhi, CHEN Chang-Qing

1998, 13(4): 333

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.

High-level expression of HIV-1 gp41 gene and purification of its product

BI Lan, LEI Xiao-Jun, CHEN Wei, YAN Jia-Xin, YU Mo-Song, SHU Jia-Hong

1998, 13(4): 344

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.

Bioassay of the multiple Autograph californica nucleopolyhedrovirus (AcMNPV) pesticides

HOU Jian-Wen, ZHAO Ye-Feng, TAO Guo-Qiang, JIANG Wei-Min, YANG Wen-Qin

1998, 13(4): 350

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

The Screen of Inhibitors of Grass Carp Hemorrhage Virus from a Phage Displayed Random Peptide Library

WANG Bing, KE Li-Hua, JIANG Hong, TIAN Po, LI Chuan-Zhao

1998, 13(4): 351

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.

Constructural analysis of antiviral peptide to grass carp hemorrhage virus

WANG Bing, KE Li-Hua, TIAN Po

1998, 13(4): 363

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.

Studies on apoptosis of HeLa cell induced by vaccinia virus

ZHANG Xiao-qing, LIU Yu, DING Ming-Xiao

1998, 13(4): 368

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.

Detection of HAV contamination in shellfish from Lianyungang sea water by antibody capture- PCR

ZHAO Wen-Ban, LIN Hong-Yu, LI Jia-Fu, WANG Li-Xin, GONG Hai-Ping, FANG Zhao-Yin, WANG Qiu-Gong

1998, 13(4): 372

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.