Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity

A technology of vascular endothelium and composition, which is applied in the field of human heavy chain variable region and its coding sequence and application, and can solve the problems of lack of anti-VEGF antibody and small molecular weight

Inactive Publication Date: 2012-06-06
SHANGHAI GENON BIOENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, so far there is still a lack of anti-VEGF antibodies with satisfactory effects in this field, especially antibodies with small molecular weight and no murine origin

Method used

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  • Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity
  • Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity
  • Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Cloning of heavy chain variable region

[0095] The total RNA extraction and reverse transcription of hybridoma cell V75 (CCTCC NO: C200623) were performed according to the kit instructions. Table 1 shows the PCR primers used for human immunoglobulin heavy chain variable region (VH) amplification. The PCR reaction conditions were: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 69°C for 30 seconds, extension at 72°C for 30 seconds, and 30 cycles; finally, extension at 72°C for 7 minutes. The PCR product of the expected size was recovered by 1.5% agarose gel electrophoresis and cloned into the pMD19-T vector.

[0096] Table 1 Universal primers used to amplify VH

[0097]

[0098] After primer pairing, it was determined that primer VH5-1 and primer VH3-3 paired can efficiently amplify a VH fragment of about 350bp ( figure 1 A). Insert the fragment into the pMD19-T vector to obtain the vector pMD19-T / VH, and the enzyme digest...

Embodiment 2

[0102] Example 2 Induced expression and purification of recombinant protein

[0103] Using the amplified product in Example 1 or the vector pMD19-T / VH as a template, use the VH-forward primer and VH-reverse primer shown in Table 1 (with Nde I and Hind III restriction sites, respectively) ) Perform PCR amplification. The amplified product (VH fragment) was digested with Hind III and Nde I, and ligated with the pET28a vector that was digested with Hind III and Nde I, so that the VH fragment was inserted into pET28a by Hind III and Nde I restriction sites. In the expression vector, the recombinant plasmid pET-VH is formed.

[0104] The pET-VH was transferred to the conventional E. coli BL21 recipient bacteria, and the optimal expression temperature (28℃, 32℃ and 37℃) and expression time (1h, 2h and 3h) were explored, and the rhVVH recombinant protein was expressed.

[0105] The amino acid sequence of the rhVVH recombinant protein is shown in SEQ ID NO: 11, in which positions 22-137 ar...

Embodiment 3

[0113] Example 3 Ability to bind antibodies to VEGF

[0114] The VEGF binding capacity of the recombinant rhVVH protein was carried out as follows: 0.5μg / mL human VEGF standard was coated with an ELISA plate, 100μL per well. The rhVVH was diluted in multiples, and VEGF monoclonal antibody (mAb produced by the V75 hybridoma cell line CCTCC NO: C200623, referred to as HVmAb, see Chinese patent application 200610116318.1)) and VEGF polyclonal antibody as a positive control group, pET28a empty The carrier extract served as a negative control group (NC). The dilution concentration and antibody used in each group are shown in Table 2, with 3 replicates in each group. Finally, the color was developed with OPD, the light absorption value of each sample at 490nm was measured, and the average value and variance were calculated.

[0115] Table 2 Sample dilution and antibody usage

[0116]

[0117] The results showed that although the primary antibody and enzyme-labeled antibody used in each ...

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Abstract

The invention provides a human derived heavy chain variable region possessing human vascular endothelial growth factor(VEGF) binding activity. Concretely, the invention provides an antibody single heavy chain variable region which is from fully human derived monoclonal antibody and possesses the human vascular endothelial growth factor binding activity. The single heavy chain variable region can be taken as an antibody fragment, the human VEGF binding activity of complete immunoglobulin can be reserved, the proliferation of human vascular endothelial cells (HUVEC) can be inhibited in dose dependent mode. The invention also provides a method for preparing the antibody fragment and its application.

Description

Technical field [0001] The present invention relates to the field of biology, and more specifically to a human heavy chain variable region with human Vascular Endothelial Growth Factor (hVEGF) binding activity and its coding sequence and application. Background technique [0002] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), was discovered by FerraraN et al. in 1989. VEGF has a relative molecular mass of (34~45)×10 3 The homodimer glycoprotein is one of the most important angiogenesis promoters discovered so far. It has an important influence on tumor infiltration and metastasis, and plays an extremely important role in the process of tumor angiogenesis. [0003] The VEGF coding gene is located on chromosome 6q21.3, with a full length of 14kb, containing 8 exons and 7 introns, and can form at least 5 protein isoforms such as VEGF121, VEGF165, VEGF189, VEGF206 and VEGF145. The biological activities of these types of VEGF are similar. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/63C12P21/02A61K39/395A61P35/00
Inventor 成国祥刘思国陈建泉
Owner SHANGHAI GENON BIOENG
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