Isolation of the Single Oocyst and Sequencing of ITS-1 of Eimeria intestinalis

[Objective] The aim was to establish a molecular biological method for identifying coccidium species.[Method]First,the pure species of Eimeria intestinalis was isolated by using single-oocyst isolation technique.Then,according to the 18s rDNA and 5.8s rDNA sequences of Eimeria coccidia published in GenBank,a pair of specific primers was designed and synthesized to amplify the internal transcribed spacer 1（ITS-1）.Finally,the PCR products were sent for sequencing.[Result]The pure species of E.intestinalis was isolated and the result of agarose gel electrophoresis showed that the PCR product was 434 bp,and at least 27-sporulated oocysts could be detected.[Conclusion]The research will provide a basis for accurate identification of coccidium species and strains.

Cloning and Sequence Analysis on IGF-1 Gene of Hubei White Swine

[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 （IGF-1） gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. [Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5＇-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3＇-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. [Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine.

Inference of Global HIV-1 Sequence Patterns and Preliminary FeatureAnalysis

The epidemiology of HIV-1 varies in different areas of the world, and it is possible that this complexity may leave unique footprints in the viral genome. Thus, we attempted to find significant patterns in global HIV-1 genome sequences. By applying the rule inference algorithm RIPPER (Repeated Incremental Pruning to Produce Error Reduction) to multiple sequence alignments of Env sequences from four classes of compiled datasets, we generated four sets of signature patterns. We found that these patterns were able to distinguish southeastern Asian from non- southeastern Asian sequences with 97.5% accuracy, Chinese from non-Chinese sequences with 98.3% accuracy, African from non-African sequences with 88.4% accuracy, and southern African from non-southern African sequences with 91.2% accuracy. These patterns showed different associations with subtypes and with amino acid positions. In addition, some signature patterns were characteristic of the geographic area from which the sample was taken. Amino acid features corresponding to the phylogenetic clustering of HIV-1 sequences were consistent with some of the deduced patterns. Using a combination of patterns inferred from subtypes B, C, and all subtypes chimeric with CRF01_AE worldwide, we found that signature patterns of subtype C were extremely common in some sampled countries (for example, Zambia in southern Africa), which may hint at the origin of this HIV-1 subtype and the need to pay special attention to this area of Africa. Signature patterns of subtype B sequences were associated with different countries. Even more, there are distinct patterns at single position 21 with glycine, leucine and isoleucine corresponding to subtype C, B and all possible recombination forms chimeric with CRF01_AE, which also indicate distinct geographic features. Our method widens the scope of inference of signature from geographic, genetic, and genomic viewpoints. These findings may provide a valuable reference for epidemiological research or vaccine design.

Antigen Gene Cloning and Expression of HIV-1 Toward an AIDS Vaccine Design Ⅰ.Amplification and Sequencing of HIV-1 Antigen Genes

Objective:To amplify antigen genes from patients with human immunodeficiency virus type 1 (HIV-1) in Guangdong Province for candidate AIDS vaccine design. Methods:Viral nucleic acid was isolated from 10 HIV-1 infected individuals' peripheral blood collected during 1995-2000 in Guangdong Province. The viral gag p24 gene and env gp120 gene were amplified by nested-PCR and sequenced. The homologies among HIV-1 isolates were compared with HIV-BLAST. Results: Among 10 HIV-1 isolates, nine are homologous to viruses of subtype B, and one is homologous to viruses of subtype E. Conclusion: Subtype B viruses of HIV-1 are predominantly present in Guangdong Province.

Characterization of 5'-proximal sequence of mouse GABAtransporter gene(GAT-1)

The cDNA molecule encoding the mouse GABA transporter gene (GAT-1) was used as probe for selecting GAT-1 gene from mouse genomic library. A positive clone, harboring the whole open reading frame of the GAT-1 protein and designated as MGABAT-G, was fished out from the library, the 5’ proximal region and nitron 1 were sequenced and analysed, and low homology was found in the above region between GAT-1 genes from mouse and human except some short conserved sequences. The DNA-protein interactions between DNA fragments containing the conserved sequences in the 5’ proximal region and nuclear proteins from different tissues of mouse were studied by means of gel-shift assay, and Southern-Western blot. The results indicate a possible positive-negative regulation mode controlling the expression of the mouse GAT-1 gene.

Sequence-specific recognition of HIV-1 DNA based upon nicking-assisted strand displacement amplification and triplex DNA

Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus （HIV） infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification （SDA） and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeflciency syndrome （AIDS） in the future.