[Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1].

OBJECTIVES: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1). METHODS: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo. RESULTS: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1. CONCLUSIONS: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

[Preparation of antibody against Memo protein and analysis of expression profile of Memo in mouse tissues].

AIM: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice. METHODS: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot. RESULTS: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot. CONCLUSION: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.

[An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli].

OBJECTIVE: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli. METHODS: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography. RESULTS: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded. CONCLUSION: This study may provide a basis for further study on the biological function of HBx at the protein level.

[Construction of recombinant plasmid using Neo/E Technology].

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

[Malignant transformation of NIH3T3 cells induced by ectopic expression of PC-1 gene].

OBJECTIVE: To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. METHODS: Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression. RESULTS: NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice. CONCLUSION: Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.

[Induction of protective immunity in rhesus monkey by inoculation with recombinant fusion protein of cholera toxin B subunit-multivalent epitopes of Plasmodium falciparum].

Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi.

[A novel system for characterization of the transcription activating proteins in mammalian cells].

A system used for detecting the transcriptional activating activity of the function-unknown gene products in mammalian cells was developed. Based on the plasmid pTet-Off and the eukaryotic expressing vector pCDNA3.1B(-)/myc-his, firstly, we constructed a set of recombinant plasmids namely pZHO1 (for cloning into the foreign gene fragment and as a negative control), pZHO2 (as a positive control). The system also includes the plasmids pTRE-luc (encoding the Firefly luciferase reporter gene) and pRL-TK (encoding Renilla luciferase gene as background control). To confirm the feasibility of the system, the plasmids pZHO1, pZHO2 and pZHO3 (encoding p53 transcriptional activating domain, containing 73 amino acids in its N terminal) was contransfected into such mammalian cells as C4-2, MCF-7, COS7 respectively, each with pTRE-luc and pRL-TK plasmids, the feasibility of the system was determined by comparing the relative activity of Firefly luciferase activity ratio of and Renilla in different transfecting panels. Our research result showed that the system we constructed can be used for detecting the transcriptional activating activity of the target protein molecules in mammalian cells.

[Expression of PC-1 gene in tumor tissues, cloning and analysis of its putative promoter region].

BACKGROUND & OBJECTIVE: PC-1 is a novel gene which is overexpressed in bone metastasis and androgen independent prostate cancer cell line C4-2. The objective of this study was to evaluate the expression level of PC-1 gene in multiple human tumor and normal tissues, and clone a series of putative PC-1 gene promoter regions and analyze their promoter activities. METHODS: PC-1 gene specific DNA sequence was used as probe to hybridize with total RNA from 10 pairs of tumor and normal tissues; The C4-2 cell genomic DNA was used as a template in the polymerase chain reaction to amplify PC-1 upstream regions from the translation initiation codon. The PCR product was directly cloned into the luciferase reporter vector pGL3-basic. C4-2 cells were transiently transfected with above recombinant plasmids and the putative promoter activities were analyzed by luciferase assay. RESULTS: PC-1 gene expression level is remarkable higher in multiple tumor tissues than in their matched normal tissues; there is no promoter activity in the 340 bp fragment from the upstream of initiation codon, while 1099 bp, 1337 bp, 1579 bp, 1831 bp, and 4939 bp fragments had the promoter activities. CONCLUSION: The PC-1 gene expression is specifically activated in multiple human tumor tissues, suggesting that PC-1 gene expression might be involved in cancer development. Our primary data shows that the highest promoter activity of PC-1 gene is within the 4939 bp DNA fragment and maybe there exists an enhancer element between the 1831 bp and 4939 bp area.