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Recombinant influenza virus rescue method and application of the same in tumor therapy

A recombinant virus and anti-tumor technology, applied in the biological field, can solve the problems of complex tumor mechanism and ineffective treatment

Active Publication Date: 2019-06-21
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the tumor mechanism is complex and the gene mutations are diverse. The introduction of one or two tumor suppressor genes is not enough to inhibit the growth of tumor cells, and the therapeutic effect is not obvious.

Method used

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  • Recombinant influenza virus rescue method and application of the same in tumor therapy
  • Recombinant influenza virus rescue method and application of the same in tumor therapy
  • Recombinant influenza virus rescue method and application of the same in tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, the rescue of recombinant influenza virus

[0093] 1. Construction of recombinant vector

[0094] The recombinant vector shown in sequence 2 is named pHW-PB1-HUPD1, the DNA molecule shown in the 887-3187 position of the sequence 2 is the PB1 gene; the sequence shown in the 3188-3253 position of the sequence 2 is the PTV-1 2A sequence ; The sequence shown in No. 3254-3313 of Sequence 2 is a signal peptide sequence; the sequence shown in No. 3314-3525 of Sequence 2 is a HUPD1 (HEAVY CHAIN) sequence; the sequence shown in No. 3526-3689 of Sequence 2 is PB1- PS packaging sequence; keep other sequences of the vector pHW2000 (sequence 1) unchanged to obtain a recombinant vector, which is named pHW-PB1-HUPD1. The recombinant vector is a vector obtained by inserting the DNA fragment (fusion gene A) shown in sequence 2 887-3689 between the BsmbI restriction sites of the pHW2000 vector, expressing PB1 and human PD1 heavy chain.

[0095] The recombinant vector show...

Embodiment 2

[0108] Example 2. Evaluation of the kinetics of antibody production during FLU-HUPD1 and FLU-HUPDL1 infection

[0109] Chicken embryos were inoculated with the recombinant influenza virus FLU-HUPD1 and FLU-HUPDL1 (-10PFUs) prepared in Example 1, respectively, and three 9-day-old SPF chicken embryos were selected at each time point (purchased from: Beijing Boehringer Ingel Hanweitong Biology Technology Co., Ltd.), set up PR8 (purchased from China Center for Disease Control and Prevention) control at the same time, inoculated chicken embryos with PR8 (-10PFUs), selected 3 chicken embryos at each time point, set up PBS control at the same time, and measured urine by ELISA method. The amount of anti-CTLA4 antibody present in the capsule.

[0110] The result is as figure 1 As shown, it can be seen that antibodies can be detected 2 days after inoculation with FLU-HUPD1 and FLU-HUPDL1, and reach a peak at 4-5 days. Neither the PR8 group nor the PBS group detected antibodies on the t...

Embodiment 3

[0113] Example 3 In vivo inhibitory effect of recombinant influenza virus FLU-HUPD1 and FLU-HUPDL1 on melanoma

[0114] FLU-HUPD1 and FLU-HUPDL1 prepared in Example 1 were washed with PBS (weighed NaCl8g, KCl0.2g, NaCl 2 HPO 4 12H 2 O 3.63g, KH 2 PO 4 0.24g, dissolved in 900ml double-distilled water, adjusted the pH value to 7.4 with hydrochloric acid, added water to make up to 1L, stored at room temperature for future use) and resuspended to obtain a titer of 5.5×10 5 pfu / 100 μl of FLU-HUPD1 suspension and a titer of 5.5 × 10 5 pfu / 100 μl of FLU-HUPDL1 suspension;

[0115] Influenza virus strain A / PR / 8 / 34 (purchased from the Chinese Center for Disease Control and Prevention) was resuspended in PBS to obtain a titer of 5.5×10 5 pfu / 100 μl of influenza virus strain A / PR / 8 / 34 suspension.

[0116] 1. Preparation of mouse subcutaneous tumor model

[0117] Human CD34 on NSG mice + Cell-reconstructed humanized immune system mice (provided by Weitongda Biotechnology Co., Ltd...

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Abstract

The invention discloses a recombinant influenza virus rescue method and an application of the same in tumor therapy. The method for preparing recombinant viruses that express antibodies to anti-tumorprotein or contain coding genes of fusion protein includes that single-stranded coding genes, heavy-strained coding genes an / or light-stranded coding genes of anti-tumor protein antibodies are savedby viruses to obtain recombinant viruses with single-stranded, heavy-stranded and / or light-stranded coding genes of anti-tumor protein antibodies and other transforming growth factor receptor or extracellular domain coding genes of the receptor. The recombinant viruses expressing immune checkpoint suppress antibodies are designed; the heavy and light chains encoding immune checkpoint suppress antibodies are constructed in the HA region and NA region of PR8 viruses respectively, and the recombinant oncolytic influenza viruses are constructed to express anti-pd1 anti-pd-L1 antibodies. The oncolytic recombinant influenza viruses provided can target and kill tumor cells without obvious impact on normal host cells and can be used for targeted tumor therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant influenza virus rescue method and its application in tumor treatment. Background technique [0002] Cancer is a leading cause of death worldwide, responsible for 8.8 million deaths in 2015. Globally, nearly one in six deaths is caused by cancer. The most common cancer types were: lung cancer (1.69 million deaths), liver cancer (788,000 deaths), colorectal cancer (774,000 deaths), stomach cancer (754,000 deaths) and breast cancer (571,000 deaths). Current conventional treatments mainly include surgical resection, transplantation, local ablation, transhepatic arterial chemoembolization, radiotherapy, and molecular targeted therapy. Therefore, a major problem in the treatment of cancer is how to efficiently and specifically kill tumor cells while reducing damage to normal cells and tissues. With the development of molecular biology and people's new understan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C07K16/28A61K39/395A61P35/00C12R1/93
Inventor 杨鹏辉张绍庚王希良王兆海余灵祥洪智贤孙芳
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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