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Recombinant adeno-associated virus (rAAV) vector for expressing full genes of human antibody and construction method of rAAV carrier

A technology of shuttle expression vector and whole gene, which is applied in the field of high-efficiency expression of human antibody whole gene, can solve the problems of low efficiency, low cutting efficiency, unstable expression efficiency of two genes, etc.

Inactive Publication Date: 2012-08-15
BEIJING UNIV OF TECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This construction scheme can avoid mutual interference during transcription of dual promoters, but the efficiency of gene expression downstream of IRES is much lower than that of upstream gene expression; (3) The construction strategy of fusion expression uses linker Connect the two genes, and translate a bifunctional fusion protein molecule under the control of a promoter, but for antibodies, the light and heavy chains need to be expressed separately to assemble into an IgG with complete function and three-dimensional structure
In addition, there are donor / acceptor site cleavage construction strategies, Furin cleavage construction strategies, 2A sequence self-cleavage construction strategies, etc. Due to the low in vivo cleavage efficiency of these construction strategies, the expression efficiency of the two genes is also very unstable. , and the regulatory mechanism of partial shearing is still unclear

Method used

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  • Recombinant adeno-associated virus (rAAV) vector for expressing full genes of human antibody and construction method of rAAV carrier
  • Recombinant adeno-associated virus (rAAV) vector for expressing full genes of human antibody and construction method of rAAV carrier
  • Recombinant adeno-associated virus (rAAV) vector for expressing full genes of human antibody and construction method of rAAV carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Construction steps of various expression schemes of pSNAV expression 2G12

[0017] 1. Construction steps of pSNAV expression 2G12 plasmid

[0018] (1) Remove the restriction site Sfi I in the AAV shuttle expression vector pSNAV:

[0019] The vector pSNAV was digested with Sfi I, the sticky-end restriction site was smoothed with T4 DNA polymerase, and then T4 DNA ligase was used to ligate into pSNAV without the Sfi I restriction site. (2) Insert the linker at the multiple cloning site of the vector pSNAV: In order to insert the light chain (L) and heavy chain (H) of IgG into the vector, a linker39 containing EcoR I--Not I--Sfi I was designed. The end has the sticky end with EcoR I and Bgl II restriction sites, and is connected with the vector pSNAV as pSNAV / linker39. The primers designed by linker39 are as follows:

[0020] Linker 39F: 5'...aattcataagaatgcggccgctataggccaactaggcca...3' (SEQ ID NO.1)

[0021] Linker 39R: 5'...gatctggcctagttggcctatagcggccgcat...

Embodiment 2

[0074] Example 2: Method for Quantitative Determination of Human IgG Content by Double Antibody Sandwich ELISA

[0075] (1) Coating: coated with Goat anti human kappa, UNLB (company: Southern Biotech, product number: 2070-01), 50ng / well, coated overnight at 4°C, washed three times with PBST, no coated 2x2 blank control in the lower right corner hole.

[0076] (2) Blocking: block with 5% skimmed milk powder, 100 μl / well, block for 1 hour at 37° C., and wash three times with PBST.

[0077] (3) Add standards and samples to be tested:

[0078]Add the IgG standard (Invitrogen, lot 1069920A, TEF 027102) and the sample to be tested on the same ELISA plate. The standard is serially diluted at 0.1 μg / ml for 8 serial dilutions from the first well, and two replicates are performed. wells; samples to be tested (including cell expression supernatant, mouse serum, etc.) were serially diluted in 8 dilution wells at a certain dilution, and two duplicate wells were made, incubated at 37°C fo...

Embodiment 3

[0084] Example 3: Expression of 2G12 at the cellular level using 14 double-gene construction schemes

[0085] The extracted high-purity plasmids (pSNAV / cLIH, pSNAV / cLcH, pSNAV / cLeH, pSNAV / cLhH, pSNAV / cLAcH, pSNAV / cLAeH, pSNAV / cLAhH, pSNAV / cHhL, pSNAV / cHcL, pSNAV / cHeL , pSNAV / eLcH, pSNAV / eLeH, pSNAV / eLAcH, pSNAV / eLAeH) were transiently transfected into 293T cells, and were transfected with FuGENE HD transfection reagent (Roche, Cat.No.04709705001). Determination of Human IgG Content by Double Antibody Sandwich ELISA Method (see Example 2 for the method) to detect the human IgG content of the cell supernatant after transfection for 72 hours. The results are shown in Table 1. By comparing the expression of these 14 construction schemes, pSNAV / eLcH is the best scheme for high-efficiency expression of the whole human antibody gene.

[0086] Table 1

[0087] construction plan

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Abstract

The invention relates to the field of molecular medicaments and gene therapy and specifically relates to an AAV (adeno-associated virus) vector expression system which is capable of efficiently expressing full genes of a human antibody and is constructed through an AAV2 / 1 shuttling expression vector pSNAV. The expression system not only has a good antibody expression effect at a cell level, but also realizes long-period and high-efficiency expression in a mammal body. The expression system provides a good platform for the application of the AVV vector for expressing the full genes of the antibody in the aspects of antibody therapy, high-yield cell strains and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine. The invention relates to a technical platform for expressing the whole gene of human antibody efficiently by using recombinant adeno-associated virus (rAAV) vector. The specific application will not be limited to the vector and antibody gene used in this study. Background technique [0002] Antibody (Antibody) refers to the immunoglobulin produced by the immune system of the body under antigen stimulation, which is produced by plasma cells proliferated and differentiated from B lymphocytes or memory cells, and can specifically bind to the corresponding antigen. Antibodies have strong specificity, It has the characteristics of small side effects, low immunogenicity, and single biological activity. Due to its unique characteristics, it has been rapidly applied to many fields of biology and medicine. At present, more than 30 antibody drugs have been approved for marketing in the world, mainly focusing o...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/66
Inventor 曾毅贾润清管永军余双庆周玉柏冯霞
Owner BEIJING UNIV OF TECH
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