Expression vector for animal cells including csp-b 5'-sar factor and method for producing recombinant proteins using same

An animal cell and expression vector technology, applied in the field of ld Attachment Reg, can solve the problem of not recording the comparative research content of MAR factor 2 copy and MAR factor 3 copy, etc.

Active Publication Date: 2014-05-14
CHONG KUN DONG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent describes the comparative research results of MAR factor 1 replica and MAR factor 2 replica, but does not record the comparative research content of MAR factor 2 replica and MAR factor 3 replica

Method used

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  • Expression vector for animal cells including csp-b 5'-sar factor and method for producing recombinant proteins using same
  • Expression vector for animal cells including csp-b 5'-sar factor and method for producing recombinant proteins using same
  • Expression vector for animal cells including csp-b 5'-sar factor and method for producing recombinant proteins using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Preparation of SAR / MAR Factors

[0072] Preparation of Human CSP-B 5'-SAR and 3'-SAR Factors

[0073] Human natural killer cells (natural killer cells, NK cells and ATCC CRL-2407) genomic DNA. Genomic DNA was prepared from NK cells using a DNA isolation kit (Dneasy Blood & Tissue Kit, Qiagen, Cat. No. 69504), and used as a template for SAR DNA polymerase chain reaction.

[0074] Using the genomic DNA of NK cells as a template, the polymerase chain reaction was performed using primers Cs5S300F (attct tcagc acctc cttaa ttttt ctccc; sequence number 5 in the sequence listing) and primers Cs5S300R (ccagg cagcc aaaga tcagt agttg tgttg; sequence number 6 in the sequence listing) . The conditions of 10 seconds at 98°C, 30 seconds at 60°C, and 2 minutes and 30 seconds at 72°C were repeated 35 times to perform a polymerase chain reaction.

[0075] Next, the polymerase chain reaction product was cloned into pGEM-T vector (Promega, Cat. No. A3600) to prepare pGEMT-CS...

Embodiment 2

[0079] Embodiment 2: Preparation of β-galactosidase expression vector

[0080] Preparation of pC06 vector

[0081] In order to prepare DNA containing cytomegalovirus (Cytomegalovirus, CMV) promoter, multiple cloning sites (Multiple Cloning Sites, MCS) and bovine growth hormone (Bovine Growth Hormone, BGH) polyadenylation sequence (polyadenylation sequence, pA) pC06 vector, the following experiments were performed. First, using the pcDNA3.1(-) vector (Invitrogen, Cat.No.V795-20) as a template, use the V6-F primer (aagct tggat ccgaa ttcat cgatg gccgg ccggt accct cgagc tgtgc cttct agttg ccagc; sequence 13 ) and V6-R primers (gctag ctaga gcccc agctg gttct ttccg; sequence No. 14 in the sequence listing) performed polymerase chain reaction. The conditions of 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute were repeated 30 times to perform a polymerase chain reaction. Then, the polymerase chain reaction product was cloned into a pcDNA3.3-TOPO vector (Invitrog...

Embodiment 3

[0094] Embodiment 3: Preparation of the β-galactosidase expression vector comprising SAR / MAR

[0095]Preparation of pCLS09G1t1 vector

[0096] In order to insert the CSP-B3'-SAR factor between the SbfI and PspOMI restriction endonuclease sites of the pCLS09G1 vector to prepare the pCLS09G1t1 vector, the following experiment was performed. First, using the pGEMT-CS3S.1.2.k vector as a template, use the cs3sSbfIF primer (aattc ctgca gggga tccca ttctc cttga tgtac taat; sequence number 26 in the sequence listing) and cs3sPsp1R primer (aattg ggccc gaatt caaac aactc aatag caaga aac; sequence listing 27 sequence) to perform polymerase chain reaction, thereby amplifying the CSP-B3'-SAR factor. The conditions of 10 seconds at 98°C, 30 seconds at 60°C, and 2 minutes and 30 seconds at 72°C were repeated 30 times to perform a polymerase chain reaction. Then, the polymerase chain reaction product was cloned into the Sbf I and PspOM I restriction endonuclease sites of the pCLS09G1 vector ...

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Abstract

The present invention relates to an expression vector for animal cells, comprising: (a) CSP-B (Cytotoxic Serine Protease-B) 5'-SAR (Scaffold or Matrix Attachment Region); (b) a promoter operable in animal cells; and (c) a polyadenylation sequence, and to a method for producing recombinant proteins using same. The vector of the present invention includes CSP-B 5'-SAR, and thus has the effect of overcoming the inhibition of gene expression according to the position of a foreign gene introduced into an animal cell, and significantly improving the expression rate of a target protein. The vector of the present invention effectively expresses recombinant proteins for drugs or antibodies in animal cells. The vector of the present invention and the method for producing recombinant proteins using same may be very usefully applied to the industrial mass production of drugs.

Description

technical field [0001] The present invention relates to an animal cell expression vector comprising the 5'-nuclear skeleton binding region (SAR, Scaffold Attachment Region) factor of CSP-B (Cytotoxic Serine Protease-B, cytotoxic serine protease-B) and the preparation of recombinant protein approach. Background technique [0002] If the foreign gene that encrypts the recombinant protein is introduced into animal cells such as Chinese Hamster Ovary (CHO, Chinese Hamster Ovary) cells, protein medicines for clinical treatment can be produced. [0003] The novel erythropoiesis-stimulating protein (NESP, Novel Erythropoiesis Stimulating Protein), which is a red blood cell growth factor, is also known as Darbepoetin alfa, which generates two N in natural erythropoietin -Protein pharmaceuticals that are genetically manipulated by linking sugar chain sites (Egrie and Browne, Br. J. Cancer, 84 Suppl. 1:3-10 (2001)). Novel erythropoiesis-stimulating proteins stimulate hematopoietic s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/12
CPCC12N9/6408C12N15/85C12N2830/46C12N2830/85C12N5/10C12N15/11C12N15/79C12P21/00
Inventor 高丽旭李相瑢金秀娟文昇基
Owner CHONG KUN DONG CORP
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