1998 Vol.13(2)
近几年来，基因治疗研究发展迅速，目的是将外源基因导入靶细胞，在体内产生疗效。早在１９９０年美国ＮＩＨ的研究人员就将腺苷脱氢酶（ａｄｅｎｏｓｉｎｅｄｅａｍｉｎａｓｅ，ＡＤＡ）基因导入病人的淋巴细胞，改善了由于ＡＤＡ基因缺损造成的免疫缺陷.
The X gene of a known adr subtype of hepatitis B virus (HBV) was cloned into the ProEX HT Prokaryotic Expression System (GibcoBRL),and the X protein of HBV,154 amino acid residues,was expressed in a fusing fashion in E.coli . The fusion protein,named His tagged X protein (shortly HX protein) contains 179 amino acid residues and its molecular weight is about 19.7 kilodaltons.It is easy to purify by the HisTrap affinity chromatography. The purity of the HX protein could reach 93%by one step of capturation.Western blotting showed that the HX protein could specifically be recognized by the human antibodies against HBV X antigen(ptotein).
With the technique of immune electron microscope,the morphology and structure of HCV were studied.After homonization of liver tissue,immune precipation with McAb to HCV C region′s antigen, and negative staining,a kind of HCV associated virus like particles was visualized.They were mainly 55-56 nm in diameter,spherical,enveloped and uneven or smooth fringe,which are similar to Togaviradae morphologically.With indirect immuno electron microscopy,there were colloidal gold particles conjugated with the virus particles and sorrounding meteriales. With immune staining of tissue piece before embed, doubious particles like virus, particles morphologically were visualized in piles. And, with in situ embed after immune staining with HCV E region′s McAb,a kind of things,which were about 50 nm in diameter,circular and conjugated with colloidal gold inside,was visualized.
Human interleukin 18 gene was isolated from human liver tissue by reverse transcription polymerase chain reaction.hIL 18 gene was cloned into plasmid pGEM T(Easy)and pBV220.The gene was sequenced and showed no difference with that reported and expresed in E.coli in the form of inclusion bodies.The recombinant protein amounts to 27.2% of total bacterial protein.
The chitinase gene of Heliothis armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV) was localized on BamHI E, BglII E,EcoRI G,HindIII F,XbaI H,BamHI+HindIII M,and BamHI+XbaI H fragments, using the α P 32 dATP labelled recombinant plasmid harboring Choristoneura fumiferana multiple nucleocapsid nucleopolyhedrovirus (CfMNPV)DNA HindIII M which contains chitinase gene as the probe. The XbaI H fragment of HaSNPV was cloned into pTZ 19R vector for further sequencing the chitinase gene.
Nucleotide sequence of the Helicoverpa armigera single nucleocapsid nuclear polyhedrosis virus (HaSNPV) HindIII K fragment has been sequenced. The fragment comprises 3255 bp, containing 15 ORFs that are at least 40 amino acids in length, including 489 bp of the 3′end of polyhedrin(ph) coding region and complete coding region (801bp) of the protein kinase (HavPk). An open reading frame, ORF 1236, encoding a 412 amino acid polypeptide was indentified between immediately downstream ph gene and upstream HavPK gene, but the transcriptional direction was opposite to them. Comparison of the nucleotide sequences and deduced amino acid sequences demonstrated that the ORF 1236 is closely similar to the ORF8 of H.zea single nuclear
polyhedrosis virus (HzSNPV),with 95.9% amino acid identity, but is distantly related to the ORF 1629 of Autographa
californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV),with only 24.8% amino acid identity.
The coat protein (CP) gene and 3′noncoding region of tomato mosaic virus Sl isolate (ToMV Sl) were amplified by RT PCR with specific primers designed on the basis of reported ToMV L strain (ToMV L) sequence. The cDNA fragment was cloned and sequenced,its sequence showed that the CP gene has 480 nucleotides in length, which encodes 158 amino acids and 3′ noncoding region contains 202 nucleotides. The nucleotide sequence of ToMV Sl showed 99.5% homology when compared with that of ToMV L and 76.2% homology when compared with that of TMV Ul. The CP gene was inserted into pGEMEX 1, Western immuno blot showed that this gene was expressed in E.coli with the proper reading frame. The gene was also inserted into plant expression vector pBI121.
According to segment 3 sequence of group B rotavirus (GBRV)IDIR strain the 23 bp oligonucleotide was synthesized and labelled with biotin.The probe was purified by HPTLC.Its sensitivity is up to 10pg by a colorimetric reaction.The probe can detect at least 100pg target seuqence of GBRV and does not hybridize to Group A and Group C rotavirus.The results showed that the probe has high specificity and sensitivity.It can be used to detect and identify GBRV,and also to research the genetic relativity of GBRV gene mutation and rearrangement.
The nucleotide sequence and location of the L4 nonstructural 100 K protein gene of the egg drop syndrome virus (EDSV),a strain AA 2 previously isolated from China,were determined.The 100K protein gene located at 55.7-64.8 m.u.has a length of 2091 nt and codes for a polypeptide of 696 amino acids (aa)with a molecular weight of 77.7kD. Comparison of the amino acid sequence of the 100K proteins from human adenoviruses and fowl adenoviruses of group I revealed a homology from 32.3% to 34.4%.Remarkably,EDSV 100K protein shares high homology (56.4% on amino acid level)with that of ovine adenovirus.
Epitope of HGV NS3 protein was analyzed by Goldkey Program,and according to predicted results,primers of NS3 gene were synthesized.HGV RNA was extracted from patients of post transfusion hepatitis,cDNA fragment of NS3 was amplified by RT PCR.Target gene was cloned into pGEM T vector,and sequence analysis showed that the fragment of NS3 is 591bp,G+C is 58.37%.The homology of nucleotide and reduced amino acid between the isolate and other strains from America and China is 84.26%-86.13%,94.42%-94.92%,respectively.The homology between Chinese strains is higher than that between American.
Two kinds of virus isolates P 14 and P a 1,P 33 were isolated from the mosaic,crinkled and stunted petunia (Petunia hybrida Vila) plants.The isolate P 14 was identified as cucumber mosaic virus (CMV) and the isolates P a 1 and P 33 as turnip mosaic virus (TuMV).CMV was the prevailing virus in petunia. Virus free research results showed as follows:(1)CMV free plantlets
were not obtained when the leaves,flower buds and meristem tips (more than 0.3mm) were cultured; (2) CMV free plantlets were gained when 0.15-0.25 mm meristem tips from the infected plants or 0.3-0.5 mm meristem tips from the infected plants after different heat treatments were cultured.
A method for analysis of fatty acids in the insect inclusion body viruses by capillary gas chromatography was described. The main components of the fatty acid chromatogram were identified ,showing that the fatty acids in the granulosis virus and nuclear polyhedrosis virus were mainly composed of undecanoic a.,dodecanoic a.,tridecanoic a.,tetradecanoic a.,hexadecanoic a., heptadecanoic a., octadecanoic a., eicosanoic a. and octadecanoic a..Based on the ratios of the related fatty acid peak heights among distinct chromatograms,the viruses were differentiated and identified.The conclusion was consistent with that of the virus monosaccharide and pyrolysis gas chromatography.
Five hybridoma cell lines secreting monoclonal antibodies(McAb) against hepatitis G virus NS 5 protein (HGV NS5) were raised by fusing Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant HGV NS5 protein. The results of indirect ELISA demonstrated that these McAbs have no cross reaction with other antigenes, such as HBsAg, HCV C,HCV NS3 protein and E.coli lysate. The titers of hybridoma culture supernatant and ascitic fluid of mice were 1.28×10 2 -2.56×10 2 ,and 6.4×10 3 -1.28×10 5 ,respectively. These monoclonal antibodies could be used in histochemistry and diagnosis of HGV infection.
Sensitive and specific anti HCV diagnostic kits are very important for preventing of the transmission of hepatitis C virus. Adding HCV NS5A antigen in diagnostic kits may increase the sensitivity of diagnostic reagents. The result of sequencing of NS5A cDNA fragment showed that it shares over 90% nucleotide acid and amino acid homology with HCV BK etc., so the isolate of
this study belongs to HCV II. Besides, a mutation at 411nt in NS5A segment, which created a stop codon TGA in the reading frame was found, indicating that the isolate might be a defective virus. Perhaps the defective virus causes the persistent infection of HCV.
