鹦鹉源I型禽副粘病毒F和HN基因序列测定与分析

Virion RNA was abstracted from purified type I Avian paramyxovirus strain YN-PA01(isolated from parrot)and used as a template. The fragment containing the fusion gene(F) and hemagglutinin-neuraminidase gene(HN) of the isolate was amplified by RT-PCR and cloned to the pMD18-T Vector. Using primer walking method the complete sequence of F-HN genes was obtained finally.And the respective amino acid sequence was deduced. Through relative software the phylogenetic trees on F gene and HN gene were constructed between strain YN-PA01 and reference strains. The results showed that strain YN-PA01 comparing with reference strains displays 98.7%-83.2% nucleotide homology and 98.1%-86.2% amino acid homology on F gene; 97.4%-79.1% nucleotide homology and 97.2%-83.2% amino acid homology on HN gene. Additional 6 amino acids are encoded by the HN gene ORF of strain YN-PA01 comparing with national reference strains.The studied strain YN-PA01 exhibits highest identities with strain JS/5[O1/Go either analyzed on F gene or HN gene.     

减蛋综合征病毒四川分离株六邻体蛋白基因的序列分析

According to the nucleotide sequence of egg drop syndrome virus(EDSV)AV-127 strain (a foreign standard isolate)from the GenBank,a pair of oligonucleotide primers was designed and synthesizedWith use of polymerase chain reaction(PCR),the hexon-encoding gene fragment was amplified from the genome of EDSV SG9301 strain that was isolated from Sichuan Province of China and the amplified fragment was directly inserted into pMD-T vectorThe recombinant plasmid harboring the hexon-encoding gene was identified by digestion of restriction endonucleases and PCRThen,a positive clone was sequencedThe result showed that the recombinant plasmid contained the complete sequence of the hexon-encoding gene and the hexon-encoding gene was 2733 base pair long which encoded 910 amino acids, identical with AV-127 strain and AAV-2 strain (an attenuated strain)Comparison with AV-127 strain indicated that there was 9985% homology at the nucleotide levelThe homology of the deduced amino acid sequence with AV-127 strain and AAv-2 strain was over 98%,which indicated that the hexon protein was conservativeBut comparatively,the homology of the hexon protein of SG9301 strain with AV-127 strain was higher(9978%),the homology of the hexon protein of AAV-2 strain with AV-127 strain and SG9301 strain were 9868% and 9846% respectivelyComparison of nucleotides and amino acids suggested that the biological character is consistent with the homologies of nucleotide and amino acid of the hexon(Namely,the homology of the hexon of virulence strain with virulence strain was higher than that of virulence strain with attenuated strain),the hexon was one of the most conserved viral proteins among EDSV strains     

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses （enJSRV）

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia （enJSRV-NM）, we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame （orf-x） that overlaps the 3＇ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain （Accession No.AF153615） and USA JSRV21 strain （Accession No. AF105220）. The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

黄瓜花叶病毒衣壳蛋白基因转化辣椒研究

The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.     

Cloning and characterization of the glutamate dehydrogenase gene inBacillus licheniformis

The gdhA genes of IRC-3 GDH strain and IRC-8 GDH strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 GDH". However, the gdhA gene from the mutant IRC-8 GDH strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I -type hexameric protein, while the GDH of Bacillus subtilis belongs to family II.     

Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens

CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal,putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites.Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1 st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells.These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.     

马铃薯Y病毒N株HC-pro基因的序列分析与表达

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 homologyof 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 .     

Different quasispecies with great mutations hide in the same subgroup J field strain of avian leukosis virus

Blood samples were collected from a local strain of chickens associated with serious tumor cases in Shandong Province.The samples were inoculated into chicken embryo fibroblast and DF-1 cells for virus isolation and identification,respectively.The inoculated cells were screened for three common chicken tumor viruses.Nine strains of avian leukosis virus subgroup J(ALV-J) were identified,and were designated LY1201‐LY1209.The env gene from the LY1201 strain was amplified and cloned.All nine resultant env clones(clones 01-09) were sequenced,and the gp85 and gp37 amino acid regions were subjected to homology analysis.Clones 01 and 03 had 10 amino acid deletions in the gp85 region compared to the other seven clones,suggesting that at least two quasispecies with obvious mutations coexist in the same field strain.Among these nine clones,three had identical gp85 and gp37 sequences,and were recognized as the dominant LY1201 quasispecies.The amino acid sequence homology of gp37 and gp85 among the nine clones was 98.5%-100.0% and 96.6%-100.0% respectively,suggesting that the gp85 region of the env gene can better display the quasispecies diversity of ALV-J than gp37.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.