Oncolytic measles virus
A measles virus and oncolytic technology, which is applied in the field of preparing the recombinant measles virus required in this article, can solve the problems of increasing cost and complicating recombinant measles virus particles, and achieve the effect of reducing cost
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Embodiment 1
[0108] Example 1: MeV cDNA plasmid vector pc3MerV2 Preparation of Id-Trka (SEQ-ID NO.3)
[0109] The preparation of the measles virus cDNA vector was performed essentially as recently described (Inoue K, Shoji Y, Kurane I, lijima T, Sakai T, Morimoto K. J Virol Methods. 2003; 107:229-36; Martin A, Staeheli P, Schneider U.J Virol. 2006;80:5708-15), but with the following important modifications.
[0110] - Initially, it was not clear (i) which sequence part / fragment of the widely used cytomegalovirus (CMV) RNA polymerase II (PolII) promoter must be replaced when used to rescue and amplify recombinant measles virus vectors considered optimal and (ii) which of the different CMV viral genomes described in GenBank (M60321, M21295) should be used for the production of CMV-based minimal promoters; therefore, priming of different minimal CMV sources had to be done first Systematic analysis of subconstructs; the results of this study showed a promoter variant construct, pc3, cover...
Embodiment 2
[0134] Embodiment 2: MeV Id-SCD (Id-SCD) plasmid vector pc3MerV2 Preparation of Id-SCD (SEQ-ID NO.4)
[0135] To generate the recombinant MeV Id-SCD (Id-SCD) measles virus vector, the plasmid pUC-SCD encoding the SCD suicide fusion gene was digested with the restriction enzyme Mlu1 and the fragment containing the open reading frame of the SCD suicide fusion gene Ligated into the base vector pc3MerV2 Id-Trka (a derivative of the parental pc3 containing an optimized (shortened and modified) CMV RNA polymerase II (Pol II) promoter) that had been linearized with the restriction enzyme AscI. Correctly integrated fragments were confirmed by restriction digestion and sequencing. Infectious virus particles were successfully produced by subsequent rescue procedures.
[0136] pc3MerV2 Id-SCD (SEQ-ID NO. 4). The sequence of pc3MerV2 Id-SCD (see figure 2 ) can be described with reference to the position of the nucleotide (nt) below:
[0137] -nt 1-55: MeV leader sequence
[01...
Embodiment 3
[0157] Embodiment 3: help the preparation of plasmid (SEQ-ID NO.5 to 7).
[0158] Preparation of helper plasmids carrying N, P or L genes respectively was performed essentially as recently described (Martin A, Staeheli P, Schneider U.J Virol. 2006; 80:5708-15), but with the following important modifications:
[0159] - As a result of a systematic analysis of different minimal CMV-derived promoter constructs, the promoter variant construct pc3 was found to give the best yield both in virus rescue and virus amplification, and was also used to aid in plasmid production;
[0160] - lack of intronic sequence in the minimal pc3 CMV promoter (301nt+3nt) prevents mRNA guidance of the splicing mechanism prior to mRNA export from the nucleus; thus reduced splicing efficiency improves measles virus rescue and amplification;
[0161] - In addition, a Hepatitis Delta Virus (HDV) ribozyme purposefully incorporated at the 3' position was used to precisely process the 3' end of all transcri...
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