Construction and application of mammary specific expression vector for human transferrin

A technology of human transferrin and carrier, applied in the biological field, can solve the problems of low expression level of human transferrin, and achieve the effect of high profit, broad commercial prospects and profit improvement.

Active Publication Date: 2014-07-09
SHANGHAI TAO TAO ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a human transferrin expression level up to 5g / L in view of the deficiency of the low expression level of human transferrin in the existing method of using the mammary gland of transgenic animals to produce human transferrin The above method for producing human transferrin by animal mammary gland and the expression vector used therein

Method used

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  • Construction and application of mammary specific expression vector for human transferrin
  • Construction and application of mammary specific expression vector for human transferrin
  • Construction and application of mammary specific expression vector for human transferrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Carrier Construction

[0027] pcDNA3.1-TFN (pcDNA3.1 is a commercial eukaryotic expression vector purchased from Invitrogen, Cat. No. V795-20). TFN is a cloned small human TF gene, including hTFcDNA and intron I, the full length is about 4.1Kb, see the sequence below).

[0028] The construction process of pcDNA3.1-TFN is as follows:

[0029] Goat genomic DNA was amplified by long-fragment PCR, and the PCR primer sequences were: 5'-AAGCAGGAATCACAGCATCTAC-3'; 5'-AGGATCCTTCATGGCTCTC GAT-3'. The human TF minigene TFN cloned by PCR was digested with Apa I+Xho I, and the pcDNA3.1 vector was also digested with Apa I+Xho I. Subsequently, a ligation reaction is carried out by using ligase to obtain the pcDNA3.1-TFN vector.

[0030] pcDNA3.1-P1A3 (P1A3 is the cloned goat β-casein gene promoter, references can be found in "High-efficiency expression of human coagulation factor IX in milk of transgenic mice guided by goat β-casein gene promoter", the sequence is shown below)....

Embodiment 2

[0099] 1. Construction of four vectors

[0100] The T1 vector is: pcDNA3.1-P1A3-TF, named pcDNA3.1-P1A3-TF;

[0101] The T2 vector is: pcDNA3.1-enhancer-P1A3-TF, named pcDNA3.1-P1A3-ETF;

[0102] The T3 vector is: pcDNA3.1-P1A3-TF-intronI, named pcDNA3.1-P1A3-TFN;

[0103] The T4 vector is: pcDNA3.1-enhancer-P1A3-TF-intronI, named pcDNA3.1-P1A3-ETFN.

[0104] The schematic diagrams of the structures of the four vectors are shown in figure 2 .

[0105] The T1 vector construction process is as follows:

[0106] Human transferrin cDNA (called TF) was amplified from the human fetal liver library. Both the cDNA and the pcDNA3.1-P1A3 vector were digested with Xho I, and the expression vector pcDNA3.1-P1A3-TF was obtained after ligation.

[0107] The T2 vector construction process is as follows:

[0108] Human genomic DNA was amplified by PCR, and the primer sequences were: 5'-CCAGCCTCGGAGTCTTCCTC-3'; 5'-TCGCTCGGTGCCAGGAGCGG-3'. A 2.2Kb-long human transferrin enhancer was amp...

Embodiment 3

[0125] 1. Comparison of expression levels of different promoters

[0126] Expression of human transferrin was performed using different promoters.

[0127] RTF: rabbit transferrin gene promoter;

[0128] EP: rabbit transferrin gene promoter + rabbit transferrin enhancer;

[0129] 2EP: Rabbit transferrin gene promoter + 2 rabbit transferrin enhancers in series;

[0130] BLG: Goat β-lactoglobulin gene promoter.

[0131] The preparation, identification and detection of expression levels of transgenic mice are the same as in Example 1.

[0132] The construction process of pcDNA3.1-RTF-TFN vector is as follows:

[0133] The rabbit transferrin promoter was amplified from rabbit genomic DNA by PCR, and the PCR primer sequences were 5'-CACGCTCCGGTCCAGCAG-3'; 5'-GAGCCTCATCTTGTAGCTCA-3', and the amplified 0.25Kb rabbit TF promoter was loaded The pGEM-T vector purchased from Promega Company, and then the vector and the pSL301 vector purchased from Invitrogen Company were double-dige...

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Abstract

The invention discloses a method for producing human transferrin by utilizing mammary gland of transgenic animals and an expression vector of human transferrin. The method comprises: utilizing the vector containing a human transferrin gene expression cassette to transfer the human transferrin gene expression cassette into a mammal genome to obtain a transgenic animal and further to obtain human transferrin from milk of the transgenic animal, wherein the expression cassette successively comprises a promoter, human transferrin gene and cattle growth hormone gene terminator from 5' to 3', the promoter is goat beta-casein gene with the sequence of SEQ ID NO. 1, the sequence of human transferrin is shown by SEQ ID NO. 2, and the vector is a eukaryotic expression carrier pcDNA3.1, and the mammals do not contain human. According to the method, the concentration of human transferrin in animal milk is at least 5 g / L and is improved by 160 times or more compared with the human transferrin concentration by employing a conventional method, and therefore the method has great business utilization value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of a human transferrin mammary gland-specific expression vector. Background technique [0002] Human transferrin (hTF) is a non-heme iron-binding glycoprotein that can bind iron ions tightly to it and selectively release it to specific sites to bind to specific receptors on target cells to perform physiological functions [1] , is an essential drug for the treatment of various diseases such as congenital atransferrinemia, and it also participates in the regulation of respiration, cell proliferation and immune system [2] . Studies in recent years have found that transferrin is also a very important regulatory factor for the growth of embryonic stem cells and neural stem cells. [0003] At present, human transferrin is mainly extracted and purified from plasma, which is not only cumbersome and inefficient, but also the raw material is easi...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C07K14/79A01K67/027
Inventor 曾凡一颜景斌曾溢滔
Owner SHANGHAI TAO TAO ENG
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