VEEV expression vector suitable for expressing antibody and application of VEEV expression vector

An expression vector and vector technology, applied in the field of VEEV expression vectors, can solve the problems of loss of self-replication function and loss, and achieve good application prospects, simple operation, and controllable positioning

Pending Publication Date: 2021-11-26
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And two helper RNAs of the Capsid protein gene and the E protein gene of the VEEV virus that do not have replication function but are respectively promoted by the sg promoter; they lack part of the gene sequence of the nsP4 protein and thus lose their self-replicating function

Method used

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  • VEEV expression vector suitable for expressing antibody and application of VEEV expression vector
  • VEEV expression vector suitable for expressing antibody and application of VEEV expression vector
  • VEEV expression vector suitable for expressing antibody and application of VEEV expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A VEEV expression vector suitable for IgG expression:

[0047] 1) Construction of Venezuelan equine encephalitis virus vaccine strain TC-83 replicon

[0048] The TC-83 replicon plasmid of the Venezuelan equine encephalitis virus vaccine strain used as a carrier was constructed by the Flavivirus Infection and Prevention Discipline Group of Wuhan Institute of Virology. ) and introduce AscI, PacI and MfeI of the TC-83 vaccine strain sequence (GenBank no.L01443.1), and construct pACYC177 plasmid vector through Not I and XbaI two restriction sites to obtain pACYC177-VEEV Replicon (TC - 83) vector plasmid.

[0049] 2) Add SG promoter sequence

[0050] The additional SG promoter sequence is amplified by PCR to amplify the own SG promoter sequence of VEEV-TC83 itself, and connected to the vector plasmid of pACYC177-VEEV Replica (TC-83) by PacI and Mfe enzyme digestion, constituting the first SG promoter, AscI, PacI, the second SG promoter, the form of MfeI, thus obtained the...

Embodiment 2

[0052] Construction of VEEV expression vector expressing novel coronavirus neutralizing antibody CB6:

[0053] The present invention is described by taking the construction of the novel coronavirus neutralizing antibody CB6 as an example.

[0054] According to the sequence of the new coronavirus neutralizing antibody CB6 (GenBank: MT470196.1 and MT470197.1), the heavy chain and light chain genes of CB6 were obtained by gene synthesis.

[0055] Using the Venezuelan equine encephalitis virus vaccine strain TC-83 replicon pACYC177-VEEVRepli con(TC-83)-2SG with two promoters as a vector, the heavy chain gene of CB6 was enzyme-cut and ligated into the first via AscI and PacI. After the first SG promoter, insert the heavy chain gene of CB6 into the second SG promoter through PacI and MfeI. The plasmid was identified as correct by DNA sequencing, and named as the replicon VEEV-CB6 replican (shown in SEQ ID NO.2).

[0056] figure 1 Middle A shows the schematic diagram of the clone ...

Embodiment 3

[0077] Detection of antibody expression in mice infected with VEEV-CB6-VRP

[0078] In this example, BalB / C mice were infected with VEEV-CB6-VRP intranasally, and at different times after infection, the expression of CB6 antibody in the lungs of the mice was detected

[0079] 1. Detection of SARS-CoV-2 S protein in lung tissue homogenate supernatant of mice infected with VEEV-CB6-VRP at different time points

[0080] 10-week-old female BalB / C mice, a total of 15 mice, were divided into 5 groups, 3 mice in each group; 4 groups were used as the V RP immunization group, and the mice were sacrificed at 1 / 3 / 5 / 7dpi for testing , Group 1 is the mock group, that is, the mice were immunized with the same amount of DMEM and killed at 3dpi for detection. Anesthetized with Avertin, intraperitoneal injection, 20g mice received about 400 μL of Avertin. After the mice were anesthetized, VEEV-CB6-VRP was used to infect the mice with nasal drops, 50uL, 5E5 IU. Three mice were taken every da...

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Abstract

The invention belongs to the technical field of animal vaccines, and particularly relates to a VEEV expression vector suitable for expressing an antibody and application of VEEV expression vector. According to the invention, a Venezuelan equine encephalitis virus vaccine strain TC-83 replicon without a structural protein gene is taken as a carrier of the mRNA, a second SG promoter is inserted, and the two SG promoters are used for respectively expressing a heavy chain and a light chain of the antibody, so that one complete antibody can be expressed by one mRNA. And the mRNA can be subjected to co-transfection packaging with helper RNAs (VEEV-C and VEEV-E without nsP4) to obtain the VRP for expressing the antibody, and the VRP can be delivered in a lung targeting manner through nasal administration and is effectively expressed, so that an effective protection effect is provided for mice.

Description

technical field [0001] The invention belongs to the technical field of animal vaccines, and in particular relates to a VEEV expression vector suitable for expressing antibodies and its application. Background technique [0002] In recent years, mRNA technology has emerged as a powerful tool for immunotherapy, and the feasibility of using mRNA-encoded antibodies has been demonstrated for the treatment of viruses, toxins, and tumors. In general, the coding sequences for the heavy and light chains of mAbs are expressed from two independent mRNAs that, together with ionizable lipids and lipid-like materials, compose lipid nanoparticles (LNPs) for in vivo delivery . Due to the different lengths and expression rates of the two chains, in order to improve the folding and assembly of the whole IgG, the ratio of light to heavy chains needs to be optimized. Currently, most antibody-encoding mRNAs mainly enter cells by encapsulating LNPs. Although the LNP system is a promising intra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C07K16/10A61K39/42A61P31/14
CPCC12N15/86C07K16/10A61K39/42A61P31/14C12N2770/36143A61K2039/543A61K2039/53
Inventor 张波李晓丹李嘉琪张哲瑞张宏庆张亚南
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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