Replication-defective drug-resistant influenza virus and nucleic acid segment recombination rate detection method thereof

A replication-deficient, influenza virus technology, applied in the field of biopharmaceuticals, can solve the problems of poor effect, lack of abundance, non-replicating influenza virus stimulating immune response for a long time, etc.

Active Publication Date: 2021-12-07
PEKING UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The function of the above-mentioned non-replicating influenza virus relies on the crossover of the antigen spectrum between the non-replicating influenza virus strain and the wild-type influenza virus circulating strain, and prevents influenza virus infection by triggering the body's immune response. less effective
First, because it takes a long time for non-replicating influenza virus to elicit an immune response, it has no effect on patients who have already been infected or even have clinical symptoms
Secondly, although the non-replicating influenza virus can elicit a more comprehensive immune response through the way of endogenous antigen presentation, it has advantages in terms of cellular immune response compared with inactivated vaccines, but the antigen spectrum it provides is not as good as that of inactivated vaccines. richer
In addition, due to the wide application of anti-influenza virus drugs at present, while inhibiting the infection of influenza virus epidemic strains, it also inhibits the effect of vaccine strains.

Method used

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  • Replication-defective drug-resistant influenza virus and nucleic acid segment recombination rate detection method thereof
  • Replication-defective drug-resistant influenza virus and nucleic acid segment recombination rate detection method thereof
  • Replication-defective drug-resistant influenza virus and nucleic acid segment recombination rate detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 12

[0082] Example 1. Construction of 12 Drug-resistant Influenza Virus Strains and Evaluation of Packaging Efficiency

[0083] 1.1 According to the mutation site analysis of different types of drug resistance of different influenza viruses and the types of small molecule drug inhibitors (such as figure 1 Shown), through gene mutation and molecular cloning methods to construct drug-resistant influenza virus strains.

[0084] First, carry out point mutations of the drug-resistant sites of the NP, PA, and NA segment plasmids, and then combine different drug-resistant mutation sites in the NA segments. The specific drug-resistant mutation sites and point mutation primers of different segments are listed in Table 1. Show.

[0085] Table 1. Point mutation primers for drug-resistant mutation sites in different segments of influenza virus

[0086]

[0087] The sequences of point mutation primers for drug-resistant mutation sites in different segments of influenza virus in Table 1 ar...

Embodiment 2

[0093] Embodiment two, systematic evaluation 12 kinds of drug-resistant influenza virus strain drug resistance rates

[0094] The drug resistance rate is evaluated by IC50 value, IC50 is the half-inhibition rate, and the standard curve is S-shaped. It refers to the drug concentration corresponding to when the amount of virus in the drug-dosed group is half that of the control sample. The higher the IC50 inhibition, the worse the sensitivity of the influenza virus to the drug. Different drug resistance mutations (take A / WSN / 33(H1N1) as an example) correspond to different drugs with decreased sensitivity.

[0095] The determination method is as follows:

[0096] 2.1 Cell preparation: Take out MDCK cells from the incubator, and observe the cell state and confluence under a microscope. Digest the cells when they are approximately 90% confluent. After aspirating the cell culture medium, add 2mL PBS buffer to wash once, add 1mL 0.25% Trypsin-EDTA, put it in the incubator and let ...

Embodiment 3

[0102] Example 3. Real-time visual labeling of five different proteins of influenza virus

[0103]Through the transpeptidase reaction, using the enzymatic reaction polypeptide fixed-point labeling technology, by inserting small short peptide sequences into the viral genome, using the corresponding enzymes to specifically recognize short peptide sequences and dyes, without affecting the growth kinetics of the virus Under the premise, fluorescent labeling of different proteins of influenza virus is realized. 5 proteins of influenza virus were labeled by 3 kinds of protein site-specific labeling techniques. The labeled influenza virus can be used to monitor the life activity of the virus after entering the cell. Specifically insert the corresponding peptide sequence on five different plasmid fragments of NA, HA, M1, M2, NP, NA-ybbR / M2-ybbR, NP-FlAsH / M1-FlAsH, HA 5Xgly .

[0104] The double arsenic-tetracysteine ​​labeling technique is used to realize the specific labeling of t...

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Abstract

The invention relates to a replication-defective drug-resistant influenza virus and a nucleic acid segment recombination rate detection method thereof. According to the genome sequence of drug-resistant influenza virus strains, 12 drug-resistant influenza virus strains are obtained through a molecular cloning technology, and the drug resistance rate of the drug-resistant influenza virus strains to anti-influenza drugs is evaluated. Seven drug-resistant replication-defective influenza virus strains (DRRIV) are constructed; visual labeled influenza viruses for detecting the recombination rate of each segment of the influenza virus are also constructed. NA28TAG / H274Y with the best effect is screened out. A small molecular NA inhibitor (NAIs) is used in a combined mode, so that the neutralizing effect of the NA28TAG / H274Y on the wild type influenza virus is further enhanced. A mouse model shows that the NA28TAG / H274Y and oseltamivir have a combined synergistic effect on the drug-resistant influenza virus.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a drug-resistant replication-deficient influenza virus constructed by codon recoding technology, its application in treating drug-resistant influenza, and a method for detecting recombination rates of different segments of the virus. Background technique [0002] A major mechanism of influenza A virus genetic diversity is the recombination of entire gene segments between co-infecting viruses. Recombination refers to the exchange of multiple single-stranded negative-sense RNA gene segments when multiple influenza viruses co-infect the same cell to form new progeny viruses. The new strain after recombination may have changes in virus replication, pathogenic mechanism and host range. Due to the continuous recombination of influenza virus genes, seasonal influenza is constantly changing, and the difficulty of fighting influenza viruses is also increasing. [0003] One ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12Q1/70G01N21/64C12N15/85C12N15/65C12N15/55C12N15/54C12N15/44A61K31/215A61K31/351A61K31/5383A61K31/496A61K39/145A61P31/16C12R1/93
CPCC12N7/00C12Q1/70G01N21/6428C12N15/85C12N15/65C12N15/52A61K31/215A61K31/351A61K31/5383A61K31/496A61K39/12A61P31/16C12N2760/16121C12N2760/16122C12N2760/16134A61K2300/00
Inventor 夏青郑哲涛史宁宁
Owner PEKING UNIV
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