Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same
A carrier and gene technology, applied in the field of human erythropoietin production, can solve the problems of lack of hEPO concentration and its activity, hEPO has not been commercialized, etc., and achieve the effect of superior physiological activity and good stability
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Embodiment 1
[0066] Example 1: Production of optimized vectors expressing mammary gland-specific hEPO of the invention
[0067] An optimized vector for secretion of hEPO in the mammary gland was produced according to the invention.
[0068] 1) Generate pBC1-hEPO vector
[0069] To generate the mammary gland-specific expression vector of the present invention, hEPO genomic DNA (SEQ ID NO: 1) was cloned at the position of the restriction enzyme XhoI using pBC1 (Invitrogen) with the goat beta casein promoter.
[0070] 2) Generate pBC1-hEPO-WPRE vector
[0071] In order to increase the expression of hEPO, the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) gene was inserted into the pBC1-hEPO vector. WPRE has been found to function as a regulator to ensure that mRNA is more stable and ultimately more protein is synthesized.
[0072] To ensure simultaneous transcription of EPO and mRNA, WPRE was ligated directly behind hEPO, and then the regulator was inserted into t...
Embodiment 2
[0083] Example 2: Generation of transgenic animals expressing hEPO and analysis of expressed hEPO
[0084] The following experiments were performed to confirm the physiological activity and stability profile of hEPO expressed by the mammary gland-specific hEPO-expressing transgenic mice of the present invention.
[0085] 1) Expression profile analysis of genes involved in glycosylation in mouse mammary gland
[0086] Glycosylation has been found to play a major role in enhancing protein function as part of post-translational modifications. Different glycosylation profiles are found in prokaryotes (including E. coli), yeast, animal cells, and mice.
[0087] Laboratory animals that have undergone stages of evolution similar to humans are thought to develop levels of glycosylation similar to those found in humans. Thus, the expression of glycosyltransferases involved in the glycosylation of recombinant hEPO was identified in the mammary gland and liver of mice and in CHO cell l...
Embodiment 3
[0157] Since this increase in physiological activity was also observed with recombinant human erythropoietin alpha treatment, we found that the in vivo activity of EPO in milk from the transgenic mice of the present invention was better. Example 3: Production of transgenic pigs using somatic cells by introducing the EPO expression vector of the present invention
[0158] Transgenic pigs were produced by introducing the expression vector pBC1 / hEPO / NEO of the present invention of Example 1 using somatic cells.
[0159] A) Prepare culture medium
[0160] In vitro maturation of follicular eggs was performed using NCSU 23 medium. 1 L of water from Baxter (Baxter Healthcare Co., U.S.A.) was filtered with a 0.2 μm filter membrane (Gelman Sci., U.S.A.), the pH was adjusted to 7.2-7.3, and then the filtrate was poured into a 50 mL tissue culture flask (Falcon, U.S.A. ), 45mL per bottle, stored at 4°C, usable for two weeks.
[0161] In vitro maturation medium was prepared using NCS...
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