丙型肝炎病毒NS3区7个抗原表位融合基因载体的构建及在真核细胞的表达

目的建立丙型肝炎病毒非结构蛋白3(NS3)区7个辅助T细胞抗原表位融合基因的真核表达载体,并在真核细胞中表达产物,为进一步研究应用HCVNS3序列进行预防HCV感染的DNA免疫的研究创造条件。方法合成3对相互重叠的寡核苷酸引物,涵盖NS3区7个辅助T细胞抗原表位,细胞通过重叠延伸PCR方法,将它们拼接在一起构建了一个多肽融合基因,经克隆测序后,插入真核表达载体pEGFPN3和pBuDCE中,用构建的pEGFPDR4质粒分别转染293T和B淋巴细胞系046W,用pBuDCEDR4质粒转染293T细胞,用Western印迹和流式细胞仪检测其表达。结果经测序证实成功的将丙型肝炎病毒NS3区7个辅助T细胞抗原表位基因序列拼接成融合基因,构建的pEGFPDR4质粒在293T和B淋巴细胞系046W中均表达了预期的融合蛋白36kD。构建的pBuDCEDR4质粒在293T细胞中可观察到预期的13kD的多肽融合蛋白。结论本研究建立了能够在真核细胞表达HCVNS3区7个辅助T细胞抗原表位融合基因的细胞系。

Construction of eukaryotic expression plasmid containing HCV NS3 segment and protein expression in human HL-7702 hepatocytes

AIM： To construct the eukaryotic expression plasmid containing HCV NS3 segment and to analyze the expression of NS3 protein in normal human hepatocyte HL-7702.METHODS： We amplified HCV NS3 fragment from plasmid pBRTM/HCV 1-3011 containing the whole length of HCV genome, recombined it with expression vector pcDNA3.1（-） to form the eukaryotic expression vector pcDNA3.1（-）/NS3, and transfected human HL-7702 hepatocytes with the recombined plasmid by cationic polymers. The expressed HCV NS3 protein was detected and analyzed by immunohistochemical method and Western blot.RESULTS： The amplified NS3 fragments had correct molecule weight and sequence. The successfully constructed eukaryotic expression plasmids were transfected to HL-7702 cells. The expressed NS3 proteins had correct molecular weight 70000.CONCLUSION： Eukaryotic expression vector pcDNA3.1 （-）/NS3 containing NS3 segment of HCV can be constructed, the sequence of NS3 fragments is consistent with the template. Normal human HL-7702 hepatocytes can efficiently express specific HCV NS3 protein in vitro.

Construction of eukaryotic expression vector carrying human TSLC1 gene and its expression in HepG2 cells

Objective： To construct the eukaryotic expression vector for human TSLC1 gene, and to express TSLC1 in HepG2 cells for investigating its effect on HepG2 cell growth. Methods： Full length of TSLC1 cDNA was amplified from RNA of normal human liver by RT-PCR, and cloned into pCI-neo expression vector. The recombinant plasmid pCI-TSLC1 was identified with restriction enzyme and sequenced, and then was stably transfected into HepG2 cells with lipofectamine 2000. The positive clones were examined by western-blotting and immunofluorescence, cell growth was analyzed with MTT assay. Results： The eukaryotic expression vector pCI-TSLC1 was successfully constructed and the stable cell line highly expressing TSLC1 protein was obtained. The growth of TSLCl-transfected cells was significantly suppressed in vitro. Conclusion： The HepG2 stable cell line could highly express TSLC1 protein, which provided a basis for further exploring the roles of TSLC1 in hepatocellular carcinoma.

EXPRESSION OF HUMAN BETA-DEFENSIN 3 IN COS-7 CELL

To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.

Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression

In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.

The Construction of the Eukaryotic Expression Vector of Glycerophosphodiester Phosphodiesterase Gene from Treponema pallidum and its Expression in Hela Cells

Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.

Archaeal chromatin proteins

Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histories, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins （e.g., archaeal histones, Sacl0b homologs, Sul7d, Cren7, CC1, etc.） have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investiga- tion of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.