CN1737136A - Production method of animal use recombinant interferon - Google Patents

Abstract

The invention discloses a production method of animal use recombinant interferon, wherein the raw recombinant interferon is subject to condensation, separating, purifying, formulation and filter membrane degerming. The method has the advantages of low cost of production, prevention of potential exogenesis viruses and other pathogenic microorganisms, and improved product purity.

Description

The production method of animal use recombinant interferon

Technical field:

The present invention relates to a kind of production method of Interferon, rabbit, the production method of especially a kind of purity height, active strong animal use recombinant interferon.

Background technology:

Interferon, rabbit is the class protein with antiviral, antitumor and immunoloregulation function that multiple inductor (as virus, bacterium and other macromole etc.) inducing cell produces.The production method of Interferon, rabbit was to be that raw material is made with animal blood in the past, cost height not only, and also have potential danger of carrying exogenous virus and other pathogenic micro-organisms in the product.In order to overcome the disadvantage that conventional production methods is brought, people have adopted genetically engineered production recombinant interferon, especially with the gene clone of animal interferon in expression vector pBV220, and be transformed in the bacillus coli DH 5 alpha, the positive colony of screening produced bacterial classification with temperature-induced expression and make the production method of recombinant interferon more advanced, its exogenous gene expression amount can reach the 20%-30% of total protein.Compare production cost with blood product and reduce greatly, can descend about 10,000 times, also avoided potential danger of carrying exogenous virus and other pathogenic micro-organisms simultaneously fully.But the activeconstituents of the recombinant interferon that existing genetic engineering process for preparing is produced all exists with the form of inclusion body, reduced the product activity effect, and product purity is not high, contains other foreign protein.

Summary of the invention:

The present invention is in order to solve the active low technical problem of existing in prior technology, and the production method of a kind of purity height, active strong animal use recombinant interferon is provided.

Technical solution of the present invention is: a kind of production method of animal use recombinant interferon, with the gene clone of animal interferon in expression vector pBV220, and be transformed in the bacillus coli DH 5 alpha, the positive colony of screening is produced bacterial classification and made rough Interferon, rabbit with temperature-induced expression, it is characterized in that also comprising the steps:

A. be that the hollow cellulose ultra-fine filter of 10000 relative molecular weights concentrates with rough Interferon, rabbit through the amount of damming;

B. spissated interferon solution is separated through Sephadex G50, series of strata 2cm*100cm with 20mmol/l, pH7.0 phosphate buffered saline buffer balance, separates with same buffer solution elution behind the upper prop earlier, checks through SDS-PAGE, collects the Interferon, rabbit part;

C. with the Interferon, rabbit collected again through the DE-52 column purification, behind the upper prop with contain 0.05,0.1, the 20mM/L of 0.15mol/LnaCl, pH7.0 phosphate buffer soln wash respectively, collects the elutriant that contains Interferon, rabbit, work in-process;

D. work in-process being made the content of resultant interference element with the dilution of pyrogen-free physiological saline is 1%～2%, uses 0.22 μ m aperture filter membrane degerming then, packing.

The present invention is on the basis of existing technology, to rough recombinant interferon concentrate, separation, purification, preparation and filter membrane degerming.The recombinant interferon of being produced except that have production cost low, avoid the potential advantage of carrying exogenous virus and other pathogenic micro-organisms, the whole process protein recovery is 25% simultaneously, do not contain foreign protein in the product, it is qualified to have improved the purity of product and DNA and thermal source matter content; Changed the existing way of activeconstituents, strengthened product activity, made the product activity composition really bring into play active function.

Embodiment:

Embodiment:

With the rough recombinant interferon of prior art for preparing, specific practice is as follows:

1. according to the various animal interferon gene orders of delivering on the Genbank, respectively the chicken splenic lymphocyte that stimulates from ConA, from the duck splenic lymphocyte of mitogenstimulated, extract geneome RNA, from pig liver and dog blood, extract genomic dna;

2. according to the Interferon, rabbit sequence of delivering on the Genbank, adopt a pair of oligonucleotide primer of Oligo software design, carry out the molecular modification of restriction enzyme at 5 of upstream and downstream primer ' end;

3. if the genome that extracts is RNA then adopts RT-PCR amplification interferon gene, if the genome that extracts is that DNA then adopts the pcr amplification interferon gene;

4.RT-PCR or the goal gene that pcr amplification obtains is through sequential analysis;

5. interferon gene is cloned among the expression vector pBV220, and is transformed in the bacillus coli DH 5 alpha, the positive colony temperature-induced expression of screening;

6.SDS-PAGE, Western Blot identifies expression product;

7. the cryopreservation of the Interferon, rabbit of stably express being reserved seed for planting is standby in the original strain storehouse;

8. spread out of from the original strain storehouse, freeze-drying is preserved after enlarging, or is spread out of with strain library by the previous generation manufacturing, enlarges the back freeze-drying and preserves as the manufacturing strain library.

9. spread out of bacterial classification from making for production usefulness with strain library, through gramstaining, electron microscopic examination, biochemical reaction, plasmid identify, the satisfactory bacterial classification of expression amount is as producing bacterial classification.

10. select for the bacterial classification (seed liquor) of doing fermentation usefulness from producing bacterial classification, seed liquor is done according to actual needs to expand and is gone down to posterity, as the seed liquor of jar cultivation;

11. fermentation culture;

12. collect thalline with centrifuging, thalline can be preserved 1 year down at-20 ℃;

13. with proper method cracking thalline such as Guanidinium hydrochloride or N,O-Diacetylmuramidase or high-pressure homogenization pumps;

14. cellular lysate is after centrifugal gained supernatant liquor is rough Interferon, rabbit.

Carry out by the following method again:

A. be that the hollow cellulose ultra-fine filter of 10000 relative molecular weights concentrates with rough Interferon, rabbit through the amount of damming;

B. spissated interferon solution is separated through Sephadex G50, series of strata 2cm*100cm with 20mmol/l, pH7.0 phosphate buffered saline buffer balance, separates with same buffer solution elution behind the upper prop earlier, checks through SDS-PAGE, collects the Interferon, rabbit part;

C. with the Interferon, rabbit collected again through the DE-52 column purification, behind the upper prop with contain 0.05,0.1, the 20mM/L of 0.15mol/LNaCl, pH7.0 phosphate buffer soln wash respectively, collects the elutriant that contains Interferon, rabbit, work in-process;

D. work in-process being made the resultant interference cellulose content with the dilution of pyrogen-free physiological saline is 1%～2%, uses 0.22 μ m aperture filter membrane degerming then, packing.

The work in-process calibrating:

Every batch of calibrating such as work in-process sampling carrying out titration, protein content, specific activity, high-efficient liquid phase color spectral purity, UV spectrum absorption, sterility test, pyrogen test, the result meets the requirements.

The finished product calibrating:

Finished product is through checks such as outward appearance, pH value, titration, telling test, sterility test, pyrogen test, proof tests.The whole process protein recovery is 25%, and product does not contain foreign protein, and DNA and thermal source matter content are qualified, and the result meets the requirements.

Claims (1)

1. the production method of an animal use recombinant interferon, with the gene clone of animal interferon in expression vector pBV220, and be transformed in the bacillus coli DH 5 alpha, the positive colony of screening is produced bacterial classification and made rough Interferon, rabbit with temperature-induced expression, it is characterized in that also comprising the steps:

A. be that the hollow cellulose ultra-fine filter of 10000 relative molecular weights concentrates with rough Interferon, rabbit through the amount of damming;

B. spissated interferon solution is separated through Sephadex G50, series of strata 2cm*100cm with 20mmol/l, pH7.0 phosphate buffered saline buffer balance, separates with same buffer solution elution behind the upper prop earlier, checks through SDS-PAGE, collects the Interferon, rabbit part;

C. with the Interferon, rabbit collected again through the DE-52 column purification, behind the upper prop with contain 0.05,0.1, the 20mM/L of 0.15mol/LNaCl, pH7.0 phosphate buffer soln wash respectively, collects the elutriant that contains Interferon, rabbit, work in-process;

D. work in-process are diluted with pyrogen-free physiological saline, making the resultant interference cellulose content is 1%～2%, uses 0.22 μ m aperture filter membrane degerming then, packing.