CN1280012A - Electric pulse transfer technology of IFN gene - Google Patents

Abstract

The present invention discloses a kind of new method of transferring interferon (IFN) gene into skeletal muscle cell to express IFN. The eucaryon expressing carrier of IFN gene is injected into the muscle tissue, and electric pulse is then applied to the injected area to promote IFN gene to enter the muscle cell for high-efficiency expression of IFN. The method is simple, safe, efficient, economic and practical, and thus has excellent application prospect.

Description

The electric pulse transfer technology of IFN gene

The present invention relates to new electric pulse transfer technology in a kind of genosome, realize the biosynthesis of the body internal interference element (IFN) of exogenous IFN gene mediated, be used for treatment of diseases such as tumor and hepatitis.

IFN is the protein of one group of energy inducing cell viral infection resisting.It has the virus replication of inhibition and hyperplasia, and the effect of immune cell activated and inducing cell differentiation is used for tumor treatment.Mostly the IFN medicine that uses clinically is by engineered way production at present, promptly adopts the method structure of gene recombinaton to contain the gene of IFN or the prokaryotic expression carrier of cDNA, and express and extract IFN in prokaryotic cell (as escherichia coli).This method relates to the IFN albumen of separation and Extraction trace from the inoculum of complicated component, complex manufacturing, and valuable product, and, because half-life weak point in the body of IFN needs heavy dose of repetitively administered to take effect, so the medical expense height.Simultaneously, because dosage is excessive, toxicity is also bigger.

The new method that the purpose of this invention is to provide synthetic IFN in a kind of body adopts the IFN gene to realize that by electric pulse transfer technology the oneself of IFN in the body is synthetic, and the technology cost is low, and method is easy, lasting medicine.

In order to realize this purpose, the present invention at first makes up the carrier for expression of eukaryon of the IFN gene that contains signal peptide, change this carrier over to escherichia coli, amplification is also extracted, then this carrier is injected skeletal muscle tissue, and implement electric pulse stimulation, impel the IFN gene to enter Skeletal Muscle Cell in injection areas, the exogenous IFN gene that the latter just can change over to is a template, synthetic justacrine IFN in the myocyte.

In this method, that separation and Extraction is not protein but DNA from inoculum, because the DNA stable in properties be convenient to separate and purify, thereby production technology is simple; Secondly, this method adopts electric pulse gene transfer technique, and easy, safety, transgene efficiency height can significantly improve the IFN expression; Because of lysosome in the Skeletal Muscle Cell is few, the DNA that changes over to is not easy to be degraded again, remaining time long (more than three months).In addition, used non-viral vector unconformity compares safety in chromosome.Thereby adopt skeletal muscle can realize the safe and efficient lasting expression of the IFN gene that changes over to as the target tissue of accepting the IFN gene, make that IFN is constantly replenished in the blood, thereby avoided because of half-life weak point in the body of IFN, need the defective of drug administration by injection repeatedly, also just greatly reduce patient's medical expense simultaneously.

The present invention specifically realizes with following steps: the method that 1. adopts gene recombinaton is inserted the pcD with eukaryotic promoter (as human cytomegalic inclusion disease virus promoter CMV) with the cDNA of IFN ₂In the plasmid, make up the carrier for expression of eukaryon that contains the IFN gene and (call pcD in the following text ₂/ IFN plasmid), this IFN plasmid can duplicate in prokaryotic cell, expresses IFN in eukaryotic cell.2. use IFN plasmid transfection escherichia coli, amplification and a large amount of preparation IFN plasmid.3. inject the IFN plasmid of purification to animal or human's muscular tissue.Injection volume reaches whose body weight according to need and decides, and every pin 100 ~ 1000 μ g should notice during injection that the direction of inserting needle is consistent with the direction that muscle fiber stretches.4. implementing pulse square wave acusector (field) in injection areas immediately after the injection stimulates: two electrode needle are thrust IFN plasmid injection areas, and apply square wave pulse voltage; The diameter of electrode needle is 0.6 ~ 1.2mm, and the spacing of two pins is 5 ~ 15mm, electrode needle thrust the degree of depth and entry needle to thrust the degree of depth roughly the same; Institute's pulse parameter of executing can and need be adjusted according to different bodies, and parameter commonly used is: the wide 20 ~ 60ms of ripple, and voltage 50 ~ 200v, frequency 0.5 ~ 2Hz, and can give electric-impulse stimulation successively by positive and negative two senses of current.Or inserting needle does not give electrical field stimulation outside skin, promptly on local skin surface, injection site, pacifies a surface electrode, with conducting resinl contact skin, and increases voltage to 300-500V.6. as required, can carry out IFN plasmid injection of multi-section position and electric pulse (electric field) stimulation applications, organize the transfer amount of IFN plasmid to build up muscle.

Practical application is given an example--and mice with tumor (as ehrlich ascites tumor, H ₂₂Hepatocarcinoma and S ₁₈₀Sarcoma) biosynthesis of skeletal muscle IFN: the method for employing gene recombinaton is inserted the pcD with CMV promoter with the cDNA of IFN ₂In the plasmid, be built into pcD ₂/ IFN plasmid; Use pcD ₂/ IFN transfection Escherichia coli obtains to contain pcD ₂The e. coli strains of/IFN; This bacterial strain is increased bacterium cultivate in the LB culture fluid, the method for employing alkaline lysis and ion-exchange chromatography is extracted the pcD in the thalline ₂/ IFN; Then, penetrate pcD to the back leg muscle intramuscular injection of mice with tumor ₂/ IFN, injection direction is parallel with femur as far as possible, and two leg muscles are injected 100 μ g DNA respectively, thrust two electrode needle in injection areas immediately after the injection, and apply the square-wave pulse electricity irritation.The diameter of electrode needle is 0.8mm, and the spacing of two pins is 6mm, electrode needle thrust the degree of depth and entry needle to thrust the degree of depth roughly the same, the pulse parameter of executing is: the wide 40ms of ripple, voltage 80v, frequency 1Hz, 3 electric-impulse stimulations are all given successively by positive and negative two senses of current in each injection site.After the plasmid injection next day the injected in mice position skeletal muscle promptly begin synthetic justacrine IFN, blood plasma IFN level continues to rise afterwards, peaks in about the 14th day, sustainable expression 2-3 is more than the month, and can obviously suppress H ₂₂Hepatocarcinoma and S ₁₈₀The survival rate of growth of sarcoma mice and significant prolongation ehrlich ascites tumor mice.

Claims (4)

1, a kind of being used for changes the IFN gene Skeletal Muscle Cell over to and expresses the technology of IFN, it is characterized in that: the IFN gene is injected muscular tissue, and carry out electric pulse stimulation in injection areas.

2, according to the described technology of claim 1, it is characterized in that: the IFN gene is the plasmid that contains the cDNA of IFN, and this plasmid has eukaryotic promoter in the upstream of IFN sequence, can duplicate in prokaryotic cell, expresses IFN in eukaryotic cell.

3, according to the described technology of claim 1, it is characterized in that: electric-impulse stimulation be with two electrodes place the IFN gene injection areas intramuscular or be affixed on local skin, apply the square-wave pulse electricity irritation then; The intensity of electric-impulse stimulation, frequency, ripple is wide and the desirability of kind, gene injection amount and the body IFN of the animal (comprising the people) that the visual receptor gene of parameter such as direction shifts etc. is regulated.

4, according to the described technology of claim 1, it is characterized in that: after giving electric-impulse stimulation, can promote the IFN gene in the expression of muscle and be released into blood, it can suppress the growth of tumor cell and improve antiviral ability.