ECHO virus for treating tumors
A virus and tumor technology, applied in the field of virus and tumor treatment
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Embodiment 1
[0269] Example 1: Acquisition and preparation of ECHO25 and its modified forms
[0270] 1.1 Isolation of ECHO25 from clinical samples of patients
[0271] (1) The patients' throat swabs and anal swabs are from China, Xiamen Center for Disease Control and Prevention; African green monkey kidney cells (Vero cells; Number: CCL-81 TM) is preserved by China, Xiamen University, National Engineering Technology Research Center for Diagnostic Reagents and Vaccines of Infectious Diseases, and cultured in MEM medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin.
[0272] (2) Specimen processing: Fully stir the patient's throat swab and anal swab in the specimen preservation solution to wash off the virus and virus-containing cells attached to the swab, and then store the specimen preservation solution at 4°C 4000rpm high-speed centrifugation for 30min;
[0273] (3) Inoculation and observation:
[0274] A. Spread Vero cells on a 24-well plate, and...
Embodiment 2
[0291] Example 2: In vitro anti-tumor experiments of ECHO25 and its modified forms
[0292] 2.1 Viruses and cell lines used
[0293] (1) Viruses: In this example, ECHO25-WT (SEQ ID NO: 12), ECHO25-HRV2 (SEQ ID NO: 13), ECHO25-miR133&206T (SEQ ID NO: 14), ECHO25- GM-CSF (SEQ ID NO: 15) and ECHO25-Anti-PD-1 (SEQ ID NO: 16).
[0294] (2) Cell line: human rhabdomyosarcoma cell RD ( Number: CCL-136 TM ); human colorectal cancer cell line SW480 ( Number: CCL-228 TM ) and HT-29 ( Number:HTB-38 TM ); human gastric cancer cell line AGS ( Number: CRL-1739 TM ), SGC7901 (CCTCC deposit number: GDC150), BGC823 (CCTCC deposit number: GDC151) and NCI-N87 ( Number: CRL-5822 TM ); human small cell lung cancer cell line NCI-H1417 ( Number: CRL-5869 TM ); human non-small cell lung cancer cell line SPC-A-1 (CCTCC deposit number: GDC050), NCI-H1299 ( Number: CRL-5803 TM ), NCI-H1975 ( Number: CRL-5908 TM ), A549 ( Number: CCL-185 TM ), NCI-H661 ( Number:HTB-183 TM )...
Embodiment 3
[0337] Example 3: In vivo anti-tumor experiments of ECHO25 and its modified forms
[0338] 3.1 Viruses, cell lines and experimental animals
[0339] (1) Viruses: ECHO25-WT (SEQ ID NO: 12), ECHO25-HRV2 (SEQ ID NO: 13), ECHO25-miR133&206T (SEQ ID NO: 14), ECHO25 provided in Example 1 were used in this example - GM-CSF (SEQ ID NO: 15) and ECHO25-Anti-PD-1 (SEQ ID NO: 16). Refer to Examples 2.2 and 2.3 for the methods of virus cultivation and virus titer determination, respectively.
[0340] (2) Cell lines: human glioma cell line GBM (primary tumor cell line isolated from patient tumor tissue), human endometrial cancer cell line Ishikawa (ECACC No.99040201), human prostate cancer cell line PC- 3( Number: CRL-1435 TM ) and human breast cancer cell line BcaP37 (CCTCC deposit number: GDC206). The above cells were all cultured in DMEM medium, and the medium was added with 10% fetal bovine serum, glutamine and penicillin-streptomycin double antibody. All above cells were store...
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