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Method of producing human forcing erythrogenin using transgene animal mammary gland

An erythropoietin, mammary gland technology, applied in the field of genetic engineering and transgenic animals

Inactive Publication Date: 2006-11-08
SHANGHAI GENON BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no report in the art on the production of EPO by mammary gland reactors (transgenic sheep or bovine mammary glands)

Method used

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  • Method of producing human forcing erythrogenin using transgene animal mammary gland
  • Method of producing human forcing erythrogenin using transgene animal mammary gland
  • Method of producing human forcing erythrogenin using transgene animal mammary gland

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of EPO mammary gland expression vector

[0043] The gene construction process of this embodiment is as follows: figure 2 As shown, the gene construct is a BLG-EPO fusion gene, wherein the regulatory sequence is derived from bovine BLG and EPO, and the coding sequence is derived from EPO.

[0044] Preparation of pBLG-EPO

[0045] 1. The source of each component

[0046] 1) Bovine BLG 5' flanking sequence and exon 1 non-coding sequence

[0047] According to the sequence reported by GENBANK X14710, the following primers were designed and synthesized: 5'-cggccgggggtctgctcc-3' (SEQ ID NO: 5) and 5'-ctcgagggctgcagctggggtcac-3' (SEQ ID NO: 6), using bovine genomic DNA as a template, using PCR Method (94°C, 5min; 94°C, 1min; 60°C, 45s; 72°C, 90s, 30 cycles) to amplify the BLG5' flanking sequence and the non-coding part of exon 1 (SEQ ID NO: 1). The PCR product was cloned into pGEM-Teasy vector (Promega Company) to obtain plasmid pBLG5'.

[0048] 2) ...

Embodiment 2

[0065] Example 2 Preparation of fusion gene pBLG-EPO transgenic goat

[0066] 1. Preparation of plasmid DNA for transgenesis

[0067] 1) Transform Escherichia coli DH52 with the plasmid pBLG-EPO, culture the DH52 strain containing the pBLG-EPO plasmid in liquid, and the culture volume can be 100ml-1000ml according to needs, then collect the bacteria, and prepare pBLG- by rapid extraction method or large-scale extraction method EPO (refer to the Molecular Cloning Handbook edited by Maniantis for detailed operation).

[0068] The extracted pBLG-EPO plasmid was digested with NotI, subjected to agarose gel electrophoresis, and a 6.82Kb fragment was recovered and purified with a SephaglassBabndprep (pharmacia biotech) kit for future use.

[0069] 2) Dilute the purified DNA to 2ug / ml with microinjection DNA diluent, centrifuge (12000rpm, 30min), aliquot and use for microinjection.

[0070] 2. Preparation of Transgenic Animals

[0071] The fusion gene BLG-EPO was introduced into t...

Embodiment 3

[0072] Example 3. Detection of fusion gene pBLG-EPO transgenic goat genome integration

[0073] 1. Sample Processing

[0074] For non-transgenic goats and transgenic goats to be tested, ears (less than 1 cm in length) were taken according to routine operations, and stored at -20°C for later use.

[0075] 2. Detection method

[0076] 1) Rapid extraction of genomic DNA: put sheep ears in a 1.5ml centrifuge tube, add 0.5ml LysisBuffer (4M Urea power, 10mM EDTA (pH8.0), 0.5% Sarkosyl (Sigma L5125), 0.1MTris- Cl (pH 8.0), 0.2M NaCl), 50 μl of proteinase K (10 mg / ml) were placed in a hybridization oven at 55° C. and shaken overnight. Centrifuge (14000rpm) for 5 minutes, transfer the supernatant to a clean centrifuge tube, add 1ml of absolute ethanol to mix gently, shake vigorously, use Tip to gently stir up flocculent DNA precipitate, and use 250-300μl of 0.1×TE Or resuspend the DNA in double distilled water, mix by pipetting, and store at 4°C.

[0077] 2) Digestion and electrop...

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Abstract

The present invention provides a new method for producing EPO, including: using bovine (B. taurus) lactoglobulin (BLG) 5' flanking sequence, part of exon intron sequence and 3' flanking sequence to regulate mammary gland-specific expression Sequence, human erythropoietin (EPO) full-length genomic DNA was used as the coding sequence to construct the fusion gene BLG-EPO, which can be expressed transiently in lactating goat mammary gland. The pronucleus of fertilized goat eggs was microinjected with the fusion gene, and a transgenic goat with mammary gland-specific and high-efficiency expression of human erythropoietin was obtained.

Description

technical field [0001] The present invention relates to the field of genetic engineering and transgenic animals. Specifically, the present invention relates to a carrier for regulating the specific expression of exogenous genes (such as human erythropoietin) in animal mammary gland tissue, and specifically expressing exogenous genes in animal mammary glands. genetic approach. The present invention also relates to a novel process for the production of human erythropoietin. Background technique [0002] Erythropoietin (EPO) is a multifunctional glycoprotein that can stimulate the proliferation and differentiation of red blood cells. It is mainly used clinically to treat renal anemia and is a valuable medical protein. The production method of EPO is mainly through microbial fermentation. However, the current production process has high cost, equipment investment requires more funds, and the pollution to the environment is also serious. [0003] Animal mammary gland tissue has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N15/06C12N15/12C12N15/85C12N15/90C07K14/505A01K67/027C12P21/06C07K14/47
CPCC07K14/505A01K67/0278A01K2207/15A01K2217/00A01K2217/05A01K2227/102A01K2267/01C07K14/4717C07K2319/00C12N15/8509
Inventor 成国祥陈建泉吴国祥赵建阳
Owner SHANGHAI GENON BIOENG
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