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Xiao-ling Shi

Publications and source records attributed to Xiao-ling Shi.

2 recordsLinked to original sources
PubMed2006

Promoter activity of SARS coronavirus 5' UTR sequence in eukaryotic cells.

OBJECTIVES: To investigate 5'UTR sequence in different SARS-CoV isolates, to identify the secondary structure, and to test the promoter activity of the cDNA sequence corresponding to SARS-CoV 5'UTR in eukaryotic cells. METHODS: 101 SARS-CoV 5'UTR were aligned. One typical sequence containing full 264 nt was then subjected to be predicted its secondary structure. The pGL3-5'UTR and pGL3-a-5'UTR were constructed by substitution of SV40 promoter with SARS-CoV 5'UTR cDNA or its antisense sequence. Then the recombinant plasmids were transfected into HepG2 cells and the luciferase activities were detected. A set of deletion mutant plasmids, of which pGL3-5' UTR-1, pGL3-5' UTR-2, pGL3-5'UTR-3 and pGL3-5'UTR-4 are with 3, 2, 1, and 0 residual stem-loops of 3' termini respectively,were constructed from pGL3-5'UTR and were transfected into HepG2 cells to express reporter gene luc+, with pGL3-5'UTR containing full sequence as control. The luciferase activities expressed by the plasmids were measured. And then the total RNA of the transfected cells was extracted. Subsequently, by 5' Rapid Amplication of cDNA Ends (5'RACE), the PCR product was sequenced. The luciferase expressed by pGL3-5'UTR in various cells, the lung carcinoma cell line A549, hepatoma cell line HepG2, kidney cell Vero E6, cervical cancer cell line HeLa and human umbilical vein endothelial cell line ECV304 were measured and compared with each other. RESULTS: The full sequence of the SARS-CoV 5' UTR is a 264nt, and 18 deletion mutants were found. Totally, 5 site substitutions were found in 101 5'UTR sequences. The SARS-CoV 5'UTR RNA folded to form a stable secondary structure containing four stem-loop domains. The biggest and most complex one is the stem-loop II appearing a pseudoknot. Comparing with pGL3-a-5'UTR, pGL3-5'UTR expressed luciferase obviously. Both pGL3-5'UTR containing full sequence and pGL3-5'UTR-1 containing three stem-loops of 3' termini expressed the luciferase well. However, when lost stem-loop I and II , the pGL3-5'UTR-2, pGL3-5'UTR-3 and pGL3-5'UTR-4 almost didn't express luciferase. The 56th nucleotide of SARS-CoV 5'UTR was found to be the initiation site for transcription. Transfected with expression luciferase plasmid pGL3-5' UTR in which SARS-CoV 5' UTR acts as the promoter, the luciferase could express in five cell lines in different degrees. Ranked by the luciferase activity from the highest to the lowest, the order is A549, HepG2, ECV304, HeLa and Vero E6. CONCLUSIONS: A: The 5'UTR sequences of different SARS-CoV isolates are relatively conserved, and a full sequence would form a secondary structure containing four stem-loop domains. B: The cDNA sequence corresponding to SARS-CoV 5'UTR possessed a promoter activity in eukaryotic cells. C: The promoter domain of the SARS-CoV 5'UTR contains both stem-loop I and II. D: The 56th nucleotide and its down stream TRS of SARS-CoV 5'UTR plays a key role in regulating transcription. E: Cells sourced from various tissues can provide efficient accessory factors for SARS-CoV 5'UTR sequence that acts as a promoter, and the lung-sourced cells may be the most suitable.

5' Untranslated Regions↗
PubMed2006

[Construction, expression and immunogenicity study of a chimeric MS/hIL-12 eukaryotic expression plasmid].

OBJECTIVE: To construct and express a chimeric Mtb8.4 with signal peptide (MS)/hIL12 eukaryotic expression plasmid, and to study the immunogenicity of the MS/hIL-12 chimeric genetic vaccines. METHODS: The MS/hIL-12 chimeric gene was amplified by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pCI-neo. The correct pCI-neo-MS/hIL12 (pMSI) recombinant plasmid was identified by PCR, restricted enzyme digestion and DNA sequencing. COS-7 cells were transfected with pMSI constructs by cationic liposome. After 48 hours, mRNA of the target gene was detected by RT-PCR, and hIL-12 protein in culture supernatant and cell lysates was detected by Western blot. C57BL/6N mice were vaccinated with MS/hIL-12 chimeric gene vaccine for three times at 3 week intervals. Four weeks after the final inoculation, three mice were sacrificed for measurement of the cytokine response and cytotoxic T lymphocyte (CTL) induction. RESULTS: The accuracy of plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMSI lead to transient expression of fusion proteins. The IFN-gamma and IL-2 titers were (1,521 +/- 48) ng/L and (755 +/- 41) ng/L in MS/hIL-12 chimeric gene vaccine group, (820 +/- 50) ng/L and (297 +/- 31) ng/L in MS gene vaccine group, (1,487 +/- 40) ng/L and (767 +/- 50) ng/L in BCG group, (121 +/- 16) ng/L and (62 +/- 10) ng/L in vacant vector group, and (48 +/- 16) ng/L and (32 +/- 17) ng/L in PBS group respectively. The levels of IFN-gamma and IL-2 in MS/hIL-12 chimeric gene vaccine group were higher than those of MS gene vaccine group, vacant vector group and PBS group (P < 0.01) and was similar to the BCG group (P > 0.05). The level of IL-4 in BCG group [(91 +/- 11) ng/L] increased significantly as compared to other groups (P < 0.01). When effector-cell-to-target-cell ratio (E:T ratio) were 100:1, 50:1, and 10:1 respectively, the CTL activity was 77.5%, 51.2%, 30.3% in MS/hIL-12 chimeric gene vaccine group, 56.2%, 37.8%, 11.5% in MS gene vaccine group, 28.9%, 21.4%, 9.8% in BCG group. The cytotoxicity in MS/hIL-12 chimeric gene vaccine group was higher than that of other groups (P < 0.01). CONCLUSION: When used to construct the chimeric gene vaccine, hIL-12 could improve the immunogenicity of MS gene vaccine.

Animals↗