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Recombinant adenovirus (rAdv) vector for expressing human antibody whole genome and method thereof

A recombinant adenovirus and shuttle expression vector technology, which can be used in recombinant DNA technology, genetic engineering, plant gene improvement and other directions, and can solve the problems of unclear shear regulation mechanism, low efficiency, and unstable expression efficiency of two genes.

Inactive Publication Date: 2012-09-26
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 adenovirus (rAdv) vector for expressing human antibody whole genome and method thereof
  • Recombinant adenovirus (rAdv) vector for expressing human antibody whole genome and method thereof
  • Recombinant adenovirus (rAdv) vector for expressing human antibody whole genome and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction steps of various expression schemes for pDC316 expressing the whole 2G12 gene

[0026] 1. Construction of shuttle plasmid pDC316 / cLcH and pDC316 / cLAcH

[0027] (1) Insert the linker into the multiple cloning site of the vector pDC316:

[0028] In order to facilitate the insertion of the light chain (L) and heavy chain (H) of IgG into the vector, a linker39 containing EcoRI-NotⅠ-SfiⅠ-BglⅡ was designed, and its two ends have sticky ends with EcoRI and BglⅡ restriction sites, which are compatible with the vector pDC316 The linkage is pDC316 / linker39. The primers designed by linker39 are as follows:

[0029] Linker 39F: 5'...aattcataagaatgcggccgctataggccaactaggcca...3' (SEQ ID NO.1)

[0030] Linker 39R: 5'...gatctggcctagttggcctatagcggccgcattcttatg...3' (SEQ ID NO. 2)

[0031] (2) PCR amplification of 2G12-L (EcoRI, NotⅠ), 2G12-LA (EcoRI, NotⅠ), 2G12-H (SfiⅠ, SfiⅠ) and CMV (NotⅠ, SfiⅠ). The amplified 2G12 light chain gene sequence (2G12-L) is sho...

Embodiment 2

[0060] Example 2: Method for Quantitative Determination of Human IgG Content by Double Antibody Sandwich ELISA

[0061] (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.

[0062] (2) Blocking: block with 5% skimmed milk powder, 100 μl / well, block for 1 hour at 37° C., and wash three times with PBST.

[0063] (3) Add standards and samples to be tested:

[0064] 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 f...

Embodiment 3

[0070] Example 3: Expression of 2G12 at the cellular level using 6 double-gene construction schemes

[0071] The extracted high-purity plasmids (pDC316 / eLcH, pDC316 / eLeH, pDC316 / eLAcH, pDC316 / eLAeH, pDC316 / cLcH, pDC316 / cLAcH) of the six construction schemes were transiently transfected into 293T cells, and FuGENE HD transfection reagent ( Roche, Cat.No.04709705001) transfection, the specific operation was carried out according to the instructions of FuGENE HD, and the human IgG content in the supernatant of cells transfected for 72 hours was detected by double antibody sandwich ELISA method (see Example 2 for the method). The results are shown in Table 1. By comparing the expression of these 6 construction schemes, it was found that pDC316 / eLcH is the best scheme for high-efficiency expression of the whole human antibody gene.

[0072] Table 2

[0073]

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Abstract

The invention relates to the field of molecular medicaments and gene therapy, in particular to an rAdv (recombinant adenovirus) vector expression system which expresses human antibody whole genome and is constructed by utilizing rAdv shuttle expression vector pDC316. The expression system has a good effect of expressing an antibody on the cell level and high expression in a mammal body in a short time, and can perform durable and long-time expression. The invention provides an important foundation for researches on rAdv vector expressed antibody whole gene in antibody treatment, tumor treatment and in-vivo expression of a novel antibody vaccine, and provides a new direction to searches on a novel neutral antibody vaccine in China.

Description

technical field [0001] The present invention relates to the fields of molecular medicine and gene therapy. Specifically, the rAdv vector expression system that efficiently expresses the entire human antibody gene is constructed by using the rAdv shuttle expression vector pDC316. The specific application will not be limited to the vector and antibody gene used in this study . The expression system not only has a good effect of expressing the antibody at the cell level, but also has a high expression in the mammalian body in a short period of time. The present invention expresses the whole gene of the antibody expressed by the rAdv vector in vivo for antibody therapy, tumor therapy and new antibody vaccines The research provided an important foundation, and also provided a new direction for the research of new neutralizing antibody gene vaccines in my country. Background technique [0002] Antibody (Antibody) refers to the immunoglobulin produced by the immune system of the bo...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66
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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