An oncolytic virus vaccine and its drug combined with immune cells to treat tumors
A technology for oncolytic viruses and vaccines, which is applied to anti-tumor drugs, medical raw materials derived from viruses/bacteriophages, viruses, etc. It can solve the problems of poor oncolytic effect, inability to successfully package, and ineffective improvement of effects, etc.
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Embodiment 1
[0058] Example 1: Construction and effect display of attenuated strains with site-directed mutation
[0059] 1. According to the manner in Table 1, site-directed mutation was performed on the Indiana strain of vesicular stomatitis virus, and three groups of mutated attenuated strains were obtained. The group number without gene mutation is: JBS000, as a control.
[0060] Table 1 The mutation status of each group
[0061]
[0062] The specific construction method of the attenuated strain is a conventional technique in the art, which is briefly described as follows:
[0063] (1) Construct the plasmid. Using the pVSV-XN2 plasmid as a template, the PCR method was used to introduce different mutation sites as described in Table 1. PCR was performed on the plasmid and primers for each mutation site, and then the PCR product was subjected to 1% agarose gel electrophoresis, and then the gel recovery kit was used for gel recovery to obtain plasmids with different mutations in the...
Embodiment 2
[0084] Example 2: Construction and effect display of oncolytic virus vaccine
[0085] 1. On the basis of each attenuated strain and wild-type virus prepared in Example 1, insert the NY-ESO-1 gene to construct an oncolytic virus vaccine. The construction schematic diagram is as follows Figure 5 shown. The insert fragments of each group are shown in Table 2.
[0086] Table 2 Display table of insert fragments in each group
[0087]
[0088] The specific preparation methods of JBS004-JBS007 are conventional techniques in the field, and are briefly described as follows. It should be noted that the following description does not limit JBS004 to JBS007 to be carried out according to the following method, but gives an example.
[0089] (1) Construction of attenuated strain plasmid. Artificially synthesized linking sequences with restriction enzyme sites Xho I and Mlu I, using biological technology and gene recombination technology, insert it into the non-coding between the G p...
Embodiment 3
[0102] Example 3: The pharmacokinetics and acute toxicity test results of JBS004
[0103] 1. Pharmacokinetic experiment. Select C57BL / 6 mice and subcutaneously inoculate 2×10 5 LLC cells, about 9 days after inoculation, the transplanted tumor grows to 100mm 3 Left and right, the LLC xenograft tumor model was established. Single intratumoral injection of 10 8 pfu / JBS004, tumor tissue samples were taken at 0 min (+15min), 6h, 12h, 48h, 96h, 120h and 14 days (repeat 3 times), the tissue was broken with a fully automatic grinder, and the tumor was extracted with Trizol The total RNA of the tissue is finally analyzed for the copy number of viral nucleic acid by quantitative PCR (fluorescent probe method). The result is as Figure 19 , 20 shown. The results showed that the amount of virus in the tumor reached its peak at 6 hours after infection, which was about 500 times more than the initial dose; 48 hours after infection, the amount of virus began to be lower than the init...
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