Herpes simplex virus vector, recombinant virus, host cell and pharmaceutical composition thereof
A technology of herpes simplex virus and virus vector, which is applied in the fields of medical formula, plant genetic improvement, genetic material composition, etc. It can solve the problems of low virus infection rate, slow replication and proliferation speed, etc., and achieve high infection rate, low toxicity, and dissolution powerful effect
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Embodiment 1
[0196] This example illustrates the preparation of a herpes simplex virus vector of the present invention.
[0197] (1) Collection of virus
[0198] An adult Chinese male, 37 years old, Han nationality, with a history of repeated oral herpes, without other diseases, had a typical clinical manifestation of herpes simplex virus type I recurrent infection—the sample was taken two days after herpes-like blisters appeared around the corner of the mouth.
[0199] Take a 15 ml sterile centrifuge tube, which contains 3 ml of sterile phosphate buffered saline (PBS) containing 100 μg / ml kanamycin, which contains 9 g / L of NaCl, 0.21 g / ml of liters of KH 2 PO 4 and 0.97 g / L of Na 2 HPO 4 .12H 2 O: Squeeze the herpetic vesicle with a sterile cotton swab until the light yellow secretion flows out, then dip the secretion with a cotton swab, and rinse the end of the obtained cotton swab with the secretion in the above-mentioned PBS buffer for 10 seconds. Take out the cotton swab, and us...
Embodiment 2
[0211] This example illustrates the lytic ability of the herpes simplex virus vector of the present invention to human nasopharyngeal carcinoma cells (CNE-1) with multiple occurrences in yellow race.
[0212] (1) Determination of Herpes Simplex Virus Vector Titer
[0213] With DMEM medium containing 10% fetal bovine serum, 5 × 10 5 Inoculate African green monkey kidney cells (Vero cells) (ATCC, USA) at a density of 1 cell / well, at 37°C in 5% CO 2 Cultivate in the incubator for 24 hours until each well of Vero cells is 80% full and ready for use.
[0214] Under sterile conditions, collect the supernatant culture fluid of the Vero cell culture cultured with 7 kinds of herpes simplex virus vectors shown in Table 2 of Example 1 respectively, and use serum-free DMEM medium to gradiently dilute to 1 × 10 respectively. 2 Double solution, 1×10 3 Double solution, 1×10 4 Double solution, 1×10 5 Double solution, 1×10 6 Double solution and 1×10 7 100 microliters of each dilution. ...
Embodiment 3
[0234] This example demonstrates the lytic ability of the herpes simplex virus vector of the present invention to general tumor cells.
[0235] According to the method of Example 2, human breast cancer cell MCF-7 and human colon cancer cell HT-29 were transfected at two different multiplicity of infection (MOI=0.1 and MOI=1) respectively with the herpes simplex virus vector of the present invention. and human lung adenocarcinoma cell A549 (purchased from the Second Research Institute of the Academy of Military Medical Sciences), and the number of survival tumor cells at different time points after transfection was tested, and the results are shown in Table 6.
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