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The Construction of the Double Expressing Adenovirus Vector Containing hTRAIL and LUC Gene

Author: GuoZhiYuan
Tutor: CuiJianLing
School: Hebei Medical University
Course: Medical Imaging and Nuclear Medicine
Keywords: adenovirus vector hTRAIL LUC IRES insulator
CLC: R346
Type: Master's thesis
Year: 2005
Downloads: 28
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Abstract

Objective: The adenovirus vector containing human TNF-related apoptosis inducing ligand (hTRAIL) and firefly luciferase(LUC) gene was constructed, which will be usefull for monitoring the express of hTRAIL indirectly by optical imaging in vivo. Methods: The 1700 bp of LUC gene was taken from plasmid PDC315-LUC by Bgl II and XbaI digestion; Pclon5 containing IRES gene were digested with BamHI and XbaI, the LUC gene was cloned into the downstream of the IRES gene of Pclon5 after they were ligated by T4 ligase, the resulting plasmid named as Pclon5-LUC. Pclon5-LUC was idendified by restriction endonucleases Kpn I and Xba I digestion. The IRES-LUC gene was taken from Pclon5-LUC by Bgl II and Xba I digestion and cloned into the Pclon9 digested with BamHI and Spe I, obtaining a multiple cloning site, generating Pclon9.5-LUC. Pclon9.5-LUC was identified by EcoR I and Age I digestion first, then identified by Sca I I digestion. The IRES-LUC fragment containing multiple cloning site was isolated from Pclon9.5-LUC by EcoRI and XhoI digestion, which ligated together with PDC312-CMV digested with EcoRI and Sal I, so the IRES-LUC fragment containing multiple cloning site was cloned into the site between the two insulators of PDC312-CMV, named as PDC312-CMV-LUC. PDC312-CMV-9.5-LUC was identified by Pst I digested. The PDC-humanTRAIL was digested with EcoR I and Xho I, the hTRAIL fragment was isolated and cloned into the multiple cloning site of PDC312-CMV-9.5-LUC after the latter it was digested with EcoR I and Sal I, resulting in the PDC312-CMV-L-TR. To identified whether the hTRAIL gene was inserted into PDC312-CMV-L-TR, the plasmid was digested with EcoR I and Sal I; To identified whether the sites of PDC312-CMV-L-TR was same as that expected, PDC312-CMV-L-TR was digested with BamH I and Kpn I. Using PDC312-CMV-L-TR as a template, hTRAIL and LUC fragment were amplified by PCR. PDC312-CMV-L-TR was cotransfected into 293 cells together with pPE3 by Lipofectamine 2000. The pPE3 containing all the adenovirus 5 gene besides 188-1139 bp. The resulting adenovirus vector named as AD-SG-hTRAIL-LUC. DNA of latter adenovirus vector was extracted and evaluated by PCR after plaques of virus were purificated three times. The recombination adenovirus vector was amplificated four times by infecting 293 cells. The titer of virus was measured by 50% tissue culture infective dose tissue microtitrimetry after purification. Result: The result of identification of intermediaryplasmids during the adenovirus vector construction. Pclon5-LUC was digested using the KpnI and Xba I for indentification, 1072 bp,1834 bp and 3127 bp of four bands were isolated by agarose gel electrophoresis. Pclon5-LUC has two sits of Kpn I and one site of Xba I, these sites lie in 658 bp, 1730 bp and 3564 bp respectively, sizes of fragments between three sits are 1072 bp, 1834 bp and 3127 bp, the same as the result of electrophoresis, which confirmed the plasmid Pclon5-LUC was constructed successfully. The plasmid Pclon9.5-LUC was digested using Age I and EcoR I, 2324 bp and 3259 bp bands were isolated by agarose gel electrophoresis. The size of the fragment of Pclon9.5-LUC from Age I to EcoR I is 2324 bp theoretically, it is the same as the fragment isolated; Pclon9.5-LUC was digested with Sca I and isolated 3169bp and 2414bp bands with agarose gel electrophoresis. Two cutting sites of Sca I are in the Pclon9.5-LUC, the distance of two sites is 3169bp theoretically, the result support what expected, which confirmed the plasmid Pclon9.5-LUC was constructed successfully. The plasmid PDC312-CMV-9.5-LUC was identified using Pst I digestion, the result of the agarose gel electrophoresis were 271 bp,607 bp, 2700 bp, 3519 bp bands. There are four Pst I sites in the PDC312-CMV-9.5-LUC, the sizes of the fragment between the four sites were equal with four bands of electrophoresis, which confirmed the vector PDC312-CMV-9.5-LUC was constructed successfully.PDC312-CMV-L-TR was digested by EcoR I and Sal I first, 766bp and 7353bp of bands were isolated by agarose gel electrophoresis. As the clone sites by EcoR I and Sal I located in the upstream and downstream of hTRAIL respectively, the size of the fragment of hTRAIL is 766bp theoretically, which mean the hTRAIL was inserted into the vector. PDC312-CMV-L-TR was digested by BamH I and Kpn I, resulting in 474bp, 795bp, 1052bp, 5798bp of four bands. PDC312-CMV-L-TR has two BamH I and two Kpn I clone sites and the above electrophoresis results should be right one theoretically. Using PDC312-CMV-L-TR as a template, two fragment was amplified by PCR. First using upstream primer VT140 and downstream primer VT158 of hTRAIL, a 766 bp fragment was amplified; then using the pimer VT159 and the VT160 of LUC, a 470bp fragment was amplified, which is equal to the size of core fragment of LUC. The result confirmed that the hTRAIL and the LUC genes were cloned into the vector. The identification of the adenovirus vector AD-SG-hTRAIL-LUC constructed: Using AD-SG-hTRAIL-LUC as a template, two fragment was amplified by PCR. First using upstream primer VT140 and downstream primer VT158 of hTRAIL, a 766 bp fragment was amplified, which was the same size as the size of hTRAIL; then using the primer VT159 and the VT160 of LUC, a 470bp fragment was amplified, which was equal to the size of core fragment of LUC. So the adenovirus vector containing LUC and

The Construction of the Double Expressing Adenovirus Vector Containing hTRAIL and LUC Gene

Abstract

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