KR890002417B1 - Method for expression of human ifn-r in coli - Google Patents

Abstract

Purified pKB-1000 vector contg. tac promotor, rrnBT1T2 transcription stop sequence and lac I, was digested with EcoRI and Hind III to give a skeletal DNA, which was ligated with a DNA contg. Hu-IFN-γ DNA at 16≰C and used to transform E.coli. Hu-IFN-γ DNA was obtained from pKK-Hu-IFN-γ plasmid contg. Hu-IFN-γ gene.

Description

Method for producing plasmid pKB-1000-γ giving Escherichia coli human immunointerferon (Hu-IFN-γ) production characteristics

1 is the nucleotide sequence of the Linker DNA,

2 is the nucleotide sequence of the tac promoter,

3 is a schematic diagram of the manufacturing process of the pKB-1000 vector and the manufacturing process of the pKB-1000-γ vector.

The present invention relates to a method for manufacturing human immune interferon (Hu-IFN-γ) by manipulating the recombinant interferon gene and the organic synthetic gene fragment using recombinant DNA technology.

In more detail, the present invention synthesizes a gene encoding a desired Hu-IFN-γ using transcription RNA (mRNA) isolated from human blood lymphocytes (Lymphocyte), and then synthesized by an organic method DNA fragments are used to connect to a strong expression vector, transformed into E. coli, stabilized, and purely isolate and limit expression vectors containing genes encoding Hu-IFN-γ from the transformed E. coli. The present invention relates to a method for producing plasmid pKB-1000-γ (KFCC-10227) capable of stably producing a large amount of Hu-IFN-γ by connecting to a production vector designed to be suitable for production conditions using an enzyme.

The recombinant plasmid prepared according to the present invention can be used to induce a large amount of Hu-IFN-γ from the transformed production strains by transforming E. coli and stabilizing and then separating and transforming E. coli again.

Interferon has been the subject of intensive research due to its antiviral effect since its existence was revealed by Isaac and Lindenmann (Proc. R. Soc. Biochem. 147, 258-267 (1957). The effort to continue is still going on.

Immune interferon is unstable at pH 2 compared to type I interferons, i.e., α-interferon and β-interferon, and its pI value is also on the alkaline side. It is also known to be a more effective interferon 50 to 100 times higher than α-interferon.

However, the research on Hu-IFN-γ and its use in humans have been delayed because they could not be obtained in large quantities, but became active when mass production by E. coli was possible by genetic engineering techniques.

In order to stably produce a large amount of Hu-IFN-γ in a simpler manner, lymphocytes isolated from human whole blood were treated with TPA and PHA to induce a large amount of transcriptional RNA for immune interferon. Synthesizing complementary DNA using the transcriptional RNA, enzymatically treating the complementary DNA to induce double-stranded DNA, and then using a suitable probe, a colony containing the human immunointerferon gene was identified. After culturing E. coli of the selected colonies in large quantities, Hu-IFN-γ gene DNA was isolated in large quantities and first confirmed by restriction enzyme digestion map, and the nucleotide sequence was determined according to the dideoxy DNA sequence determination method. Confirmed. Then, for efficient translation, a linker with a palindrom structure containing specific sequences and restriction enzyme positions (see FIG. 1) is synthesized and linked after a strong promoter, and the γ-interferon After transforming Escherichia coli by linking the genes correctly, the expression vector containing the gene encoding Hu-IFN-γ was isolated from the transformed Escherichia coli, and then linked to the production vector using restriction enzymes. By transforming E. coli, Hu-IFN- [gamma] was stably expressed in a large amount from the transformed production strain.

The production vector contains both the basic element as a vector and all the enzymes necessary for expressing the inserted DNA, and can produce as much protein as desired even in a well controlled range so as not to affect the stability of the vector DNA and the water. Recombinant recombinant vector (pKB-1000) was used.

Recombinant DNA technology enhances the affinity of the promoter for RNA polymerase, allowing RNA polymerase to transcribe transcriptional RNA at high frequencies, or the base sequence between the ribosome attachment site and the protein interpretation site (ATG). By controlling the distance, the protein can be more efficiently interpreted from the transcribed mRNA to produce the desired protein.

However, overproduction of unwanted products in host cells by foreign genes is likely to be detrimental to host cells. Such over-produced foreign gene products reduce the stability of the host cell vector system and may result in deletion or mutation of plasmid DNA. Through the removal of the DNA or slowing the growth rate of the host cell can be difficult to fermentation system.

Therefore, these foreign gene products must be produced in a precisely controlled state.

The expression vector pKK233-3 used by the present inventors has a tac promoter which is one of the strongest promoters and an rrnBT ₁ T ₂ structure which is an efficient transcription termination sequence. However, in order to operate this expression system in a well controlled state, E. coli having lac I ^Q mutations on chromosome DNA such as JM 101, JM 103 and JM 105 must be used as the host. In the case of using the general strain lacking, the foreign substance is continuously expressed, and the stability of the recombinant plasmid is dangerous. Therefore, since the type of E. coli that can be selected as a host is extremely limited, the present inventors and the like insert the lac I ^Q gene into pKK223-3 to give pKK223-3 its own control ability to the type of host to be used. It was possible to produce proteins in a controlled state regardless. The tac promoter is a hybrid promoter linking the -35 region of the trp promoter and the -10 region of the lac UV5 promoter. The tac promoter has a strong affinity for RNA polymerase and the lac UV5 promoter specifically induced by IPTG. RrnBT ₁ T ₂ is known as the transcription termination sequence of strong ribosomal RNA (plasmid 6,112 ('81), J. Mol. Biol. 148, 107 ('). 81) Brosius et al.).

The lacI gene has long been known as a gene that produces a repressor protein that binds to the operator of the lac gene and stops RNA polymerase that is transcribed from the lac promoter, thereby making it unable to produce any more RNA (Jeffrey H). Miller "operon" P. 81 CHS).

Since the lacI gene product binds more specifically to the compound IPTG, the addition of IPTG allows the immediately closed lac operator to be drilled, allowing RNA polymerase to pass through the lac operator region. Therefore, the transcriptional RNA of the protein of interest is produced, so that the protein of interest can be produced.

Example 1

Recombination of pKB-1000.

The pKK-223-3 plasmid was purified purely using an ultrafast centrifuge, concentrated using alcohol, and part of it was completely digested with PvuI restriction enzyme in a buffer solution containing high concentration of salt, 3.5% Polyacrylamide gel electrophoresis (PAGE) was used to isolate the DNA digested with PvuI, which was then purified and concentrated by sequentially treating with phenol, chloroform, and ethanol. Then, a portion of this was taken and reacted by changing a small amount of SalI and partial cleavage conditions from 0 to 30 minutes, and the remaining amount of DNA was partially cleaved by reacting with SalI under conditions determined to be appropriate. The cleaved DNA was separated through PAGE, and then purified and concentrated by pure separation under the conditions described above.

The pINIII.A3 plasmid was simultaneously digested with PvuI and SalI and completely cleaved, followed by PAGE to isolate the DNA band containing the lacI gene. The separated gel was treated sequentially with phenol, chloroform and ethanol to isolate and purify DNA.

As described above, the two kinds of separated, purified and concentrated DNAs are connected to each other using DNA ligase under suitable conditions to form cyclic DNA, and then treated with calcium chloride to obtain a suitable state. Transformed into E. coli.

Then, the cells were cultured in LB ampicillin medium, separated into a single colony, and then cultured in small amounts again. The transformed DNA was isolated according to the DNA minipreparation method (Molecular cloning CSH) used by T. Manniatis et al. Next, a suitable restriction enzyme was used in combination. At this time, the first screening was performed using PvuI and SalI as restriction enzymes, and then second screening was performed using EcoRI, BamHI, PstI, and HindIII in duplicate.

Example 2

Preparation of a Recombinant Plasmid (pKB-1000-IFN-γ) with Self-Controlling Effects and Characterization of Hu-IFN-γ Production.

The pKB-1000 vector containing the tac promoter, rrnBT ₁ T ₂ transcription termination sequence, and lacI gene was isolated purely, completely cleaved with EcoRI and HindIII in the presence of 100 mM NaCl, and separated by PAGE to isolate the desired DNA fragment (skeleton DNA). Purified and concentrated according to the method.

Then, the pKK-Hu-IFN-γ plasmid containing the Hu-IFN-γ gene was separated in large quantities and completely cleaved with EcoRI and HindIII under 100 mM NaCl, followed by PAGE containing Hu-IFN-γ DNA. The purified DNA was purified and concentrated according to the method described above, and further purified by passing through a G-50 column.

The above-described skeletal DNA and the DNA containing Hu-IFN-γ DNA were reacted with each other for 3 to 5 hours in a 16 ° C. water bath, and then transformed into E. coli, which was made suitable for transformation using 50 mM CaCl ₂ . Switched Transformed colonies were separated into single colonies on LB ampicillin plates, and then DNA was isolated according to the minipreparation method. Then, after mixing and digesting with EcoRI and BamHI or SalI and PvuI, the cells were first screened for colonies containing the Hu-IFN-γ DNA as intended by hanging on an agarose gel to confirm the length. The protein band generated by PAGE was analyzed and confirmed second, and finally confirmed by examining immunological specific reactions.

Example 3

Transformation of the host

Various kinds of Escherichia coli (HB 101, YMC 9, JM 101, JM 103, JM 105 and MM 294) were made in a competent state using CaCl _2, and then prepared according to the above-described method. The recombinant plasmid Hu-IFN-γ confirmed by pure separation was transformed into each. After culturing each of the transformed strains, the best strains were selected by generating Hu-IFN-γ and comparing them with or without IPTG.

The titer of the produced Hu-IFN-γ was measured by examining the cytotoxic effect of cell death using MDBK cells or WISH cells and VSV viruses, and the stability of the recombinant plasmid was determined by using ampicillin. It was cultured in a medium containing or without and recovered every 10 to 20 generations and examined by separating and analyzing plasmid DNA. Through this analysis, stable strains were selected even after 100 to 400 generations or more of culture.

Example 4

Hu-IFN-γ production control effect by lacI gene.

By using the transformed strain isolated in Example 3 to determine the effect of interfering cell death, it is determined whether the recombinant plasmid pKB-1000-IFN-γ prepared according to the method of Example 2 exhibits the self-control effect as intended. It was.

In case of using JM 101 and JM 105 with lacI ^Q mutation on chromosome DNA as host, the control ability of Hu-IFN-γ was further enhanced, and when using E. coli lacking lacI ^Q mutation as host Although there was a slight degree difference according to the cells, the production of Hu-IFN-γ was confirmed to be controlled to a remarkable degree (80-95%).

Therefore, when the foreign gene is expressed using the plasmid pKB-1000 prepared by the present inventors, it is proved that the production of the protein can be controlled regardless of the gene type of the host cell, thereby achieving stability of the recombinant strain.

Claims (2)

Linking the tac promoter of the pKB-1000 vector with the lacI gene capable of regulating the activity of the promoter in the pKK223-3 vector having the tac promoter and rrnBT ₁ T ₂ transcription termination sequence and the DNA encoding the immunointerferon using linker DNA A method for preparing a plasmid pKB-1000-γ (KFCC-10227) having human immunointerferon production characteristics, characterized by schikim. The method of claim 1, wherein the plasmid pKB-1000-γ has a specific linkage structure of 5′-AACAGAATTCT-3 ′ sequence between the ASD sequence of the pKB-1000 vector and the start codon.

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