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Human thrombopoietin expression vector and constructing method therefor

A technology of thrombopoietin and expression vector, applied in the field of genetic engineering and transgenic engineering

Inactive Publication Date: 2008-08-27
上海医学遗传研究所 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the construction of mammary gland-specific expression vectors using hTPO gene (Genomic DNA, cDNA) and goat-β casein promoter, and there is no report on the use of hTPO gene (Genomic DNA, cDNA) vectors and the production of hTPO by transgenic mammals

Method used

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  • Human thrombopoietin expression vector and constructing method therefor
  • Human thrombopoietin expression vector and constructing method therefor
  • Human thrombopoietin expression vector and constructing method therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 : Cloning and determination of hTPO genomic DNA sequence

[0036] 1. PCR amplification

[0037] Using human fetal liver genomic DNA as a template and a pair of nucleotides as primers;

[0038] Oligonucleotide A1: 5'-AGGTACCTGATGACCTGCTGCTGT-3' is the forward primer,

[0039] Oligonucleotide A2: 5'-CGGCGCTCCCATTTATTCCTTAC-3' is the reverse primer.

[0040] PCR amplification was performed using the Long-PCR Kit (Bao Biological Engineering Company). The PCR reaction conditions are: 95° C. for 1 min; (98° C., 20 sec; 65° C., 10 min)*30; 72° C., 10 min. Then electrophoresis detection of target fragments.

[0041] 2. Sequencing of PCR products

[0042] The PCR amplification products obtained above were respectively connected to pGEM-T vector (Promega Company), and transfected into Escherichia coli Top 10F' host competent cells by conventional methods, a small amount of plasmid was amplified, and the plasmid was extracted by alkaline lysis. A small amount of ...

Embodiment 2

[0049] Example 2 : Cloning and sequencing of hTPO cDNA sequence

[0050] 1. RT-PCR

[0051] Using human fetal liver mRNA as a template, first use RT-PCR Kit (STRATA GENE) to reverse, then use the cDNA obtained by reverse transcription as a template, and use two pairs of (B1 / B2, C1 / C2) nucleotides as primers

[0052] B1: 5'-GCGTCGACAGAATGGAGCTGACTGAATT-3' is the forward primer

[0053] B2: 5'-GGCATTGGGATCCTTGTGAGCTGTGG-3' is the reverse primer;

[0054] C1: 5'-CAGCTCACAAGGATCCCAATGCCATCTTC-3' is the forward primer,

[0055] C2: 5'-CGCAGCTGACACGGCAGTGTCTGAGAACCTTA-3' is the reverse primer for PCR amplification.

[0056] The reaction conditions for PCR were: 95°C for 5 min; 54°C for 5 min; (94°C for 1 min; 62°C for 1 min; 72°C for 90 sec)*35; 72°C for 10 min. Then, the target fragment was detected by electrophoresis, and two hTPO cDNA fragments of 450bp and 660bp were obtained respectively.

[0057] 2. Ligation and sequencing of two fragments of hTPO

[0058] The two cDNA...

Embodiment 3

[0065] Example 3 , Construction of mammary gland-specific expression vector

[0066] 1. The construction of the hTPO genomic DNA expression vector co-activated by the pCMV of pcDNA3.1 (-) and the 5' end 6606bp sequence of goat β-casein gene

[0067] The PCR product of hTPO genomic DNA was connected to the T vector, and loaded into the cloning vector pBSK after double digestion with Sal 1 and Sma I (NEB company); then digested with NotI and KpnI to cut out the hTPO genomic DNA, and similarly digested eukaryotic cells The gene expression vector pcDNA3.1(-), the target fragment was recovered by electrophoresis, and the above fragments were sequentially loaded into the pcDNA3.1(-) expression vector with T4 ligase (NEB company); finally, the goat was digested with NotI The 6609bp sequence at the 5' end of the β-casein gene was inserted into the 5' end of the hTPO genomic DNA, and the clones with the correct direction were selected for identification by enzyme digestion, and the j...

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Abstract

The invention belongs to the technical field of genetic engineering and transgenic engineering and in particular relates to a method for building mammary gland specific expression vectors of human thrombopoietin gene and goat beta-casein gene promoter. The vectors which are built are transferred to COS-7 and HC-11 cells and expressed on eukaryotic cell level verification hTPO. The built mammary gland specific expression vectors of the human thrombopoietin gene and the goat beta-casein gene promoter are applied to prepare a mammary gland bioreactor of transgenic animals and are expressed with high level in the milk of transgenic mammalian, and the vectors provide useful drug protein-hTPO for clinic. The mammary gland specific expression vectors of human thrombopoietin gene and goat beta-casein gene promoter which are provided by the invention provide a foundation for further research and development.

Description

[0001] This application is a divisional application with the application number 02136029.4, the filing date is July 13, 2002, and the invention title is "a preparation method of human thrombopoietin". technical field [0002] The invention belongs to the technical field of genetic engineering and transgenic engineering, and relates to a preparation method of human thrombopoietin. It specifically relates to the construction of human thrombopoietin (hTPO) genome DNA and cDNA expression vectors guided by goat-beta casein promoter and the method for producing hTPO from cells transfected with these vectors and transgenic animals. Background technique [0003] Thrombopoietin (TPO) is an important hematopoietic factor, and its receptor c-mpl is mainly found in megakaryocytes, platelets and hematopoietic precursor cells. Therefore, the main biological function of TPO is to stimulate the proliferation, differentiation and platelet formation of megakaryocytes. Maturation and accelerat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12
Inventor 曾溢滔宁云山黄淑帧王小宁
Owner 上海医学遗传研究所
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