CN1766093A - Method for preparing antibiotic peptide and transfer factor by in vitro culture and induction of lymphocyte - Google Patents

Abstract

The invention discloses a method to prepare antibacterial peptide and transfer factor by culturing and inducing lymphocyte in vitro, which comprises: preparing and subculturing lymphocyte single layer by spleen or peripheral blood; using canavaline A to induce and culture the single layer, and obtaining the product. This invention needs just supersonic wave and frost-thaw treatment without purification, can apply the product directly on animal for antibiosis and antiviral, needs not to slay lots of live pigs, has high yield with low cost, and can prevent the product bringing extraneous virus.

Description

The vitro culture induction of lymphocyte prepares the method for antibacterial peptide and transfer factor

Technical field:

The present invention relates to the production method of antibacterial peptide and transfer factor, the vitro culture induction of lymphocyte that especially a kind of production technique is simple, output capacity is high prepares the method for antibacterial peptide and transfer factor.

Background technology:

Antibacterial peptide (Anti-bacterial peptides, be called for short ABP) be through inducing, the defensive peptide class active substance that resists exogenous pathogenic agent by the generation of animal immune system of defense, molecular weight is little, activity is strong, have antibacterium, fungi, virus and protozoon effect, even cancer cells also had lethal effect, use very extensive.Present preparation method is to be raw material with the fresh pig small intestine, degrease, serous coat and content, and freezing the shredding in back of weighing smashed pulping to pieces.Water proof boiled 15 minutes after the homogenate, added 5% acetate according to 1: 1 ratio, and stirred under 4 ℃ of temperature condition and spend the night, next day with mixture under 4 ℃ of temperature condition with 8000 rev/mins centrifugal 30 minutes, get supernatant, be kept in 4 ℃ of temperature environments.Throw out is mixed with equal-volume 5% acetate, place 4 ℃ of stirring and leaching to spend the night again, repeat above-mentioned centrifugal process, get supernatant, merge supernatant liquor twice, adjust the pH value, recentrifuge is abandoned precipitation, and supernatant liquor lyophilize product are antibacterial peptide.

(Transfer factor is to have a kind of lower molecular weight polynucleotide peptide that immunocompetent lymphocytes discharges TF) to transfer factor, and it can pass to acceptor specifically with certain specific cellular immune function of donor.Present preparation method is: fresh or refrigerated spleen and lymphoglandula at 25 ℃ of left and right sides degreases and reticular tissue, are cleaned with purified water then; The tissue of cleaning is cut into small pieces, adds suitable quantity of water, use tissue mashing machine's smudge cells, make homogenate, add an amount of purified water, mixing is put-20 ℃ of multigelations 5～8 times, and melt temperature must not be above 37 ℃; With the homogenate of freeze thawing centrifugal (4 ℃ of centrifuging temperatures), go precipitation, stay supernatant liquor standby; Supernatant liquor dress dialysis tubing was dialysed 24-48 hour; The work in-process filtration sterilization; The configuration of finished product and check.

The existing method for preparing antibacterial peptide and transfer factor exists following shortcoming:

1. complex manufacturing, output capacity are low, and the antibacterial peptide that is extracted need be further purified and could obtain the purity high product, the product cost height;

2. must become raw material with fresh pig small intestine, spleen and lymph,, can only slaughter a large amount of live pigs if will obtain a large amount of antibacterial peptides, transfer factor;

3. potential danger of carrying exogenous virus and other pathogenic micro-organisms is arranged in the product.

Summary of the invention:

The present invention be for solve the product existing in prior technology complex manufacturing, output capacity is low, cost is high and raw material sources is less, potential technical problems such as danger of carrying exogenous virus and other pathogenic micro-organisms are arranged in the product, provide that a kind of production technique is simple, output capacity is high, cost is low, the method that the raw material consumption is few, vitro culture induction of lymphocyte that can avoid product to carry exogenous virus and other pathogenic micro-organisms prepares antibacterial peptide and transfer factor, and the cultivation of can repeatedly going down to posterity is produced.

Technical solution of the present invention is: a kind of vitro culture induction of lymphocyte prepares the method for antibacterial peptide and transfer factor, it is characterized in that comprising the steps:

A. prepare the lymphocyte individual layer with spleen or peripheral blood;

B. to the cultivation of going down to posterity of lymphocyte individual layer;

C. after the lymph passage cell grows up to individual layer, use the concanavalin A inducing culture.

The present invention is a raw material with less spleen or peripheral blood, makes the lymphocyte individual layer, can produce the mixture of a large amount of antibacterial peptides and transfer factor at vitro culture, induction of lymphocyte individual layer.Have following advantage:

1. need not further purification, can directly mixture be used in after involving the freeze thawing simple process on one's body the animal, can play antibiotic, antiviral effect, also can improve the immunologic function of acceptor simultaneously through ultrasonic;

2. production technique is simple, and the raw material consumption is few, need not to slaughter a large amount of live pigs, can produce a large amount of product (antibacterial peptide and transfer factor), and output capacity height and cost are low, can save great amount of manpower and material resources;

3. can avoid product to carry exogenous virus and other pathogenic micro-organisms.

4. can repeatedly go down to posterity to cultivate and produce antibacterial peptide and transfer factor.

Embodiment:

Embodiment 1:

1. be raw material with the fresh pig spleen, prepare the lymphocyte individual layer according to a conventional method.

Concrete operation method can carry out according to the following steps:

A. handle the fresh pig spleen

Get the fresh pig spleen, put into aseptic beaker immediately, the adding balanced salt solution soaks half an hour or is dipped in the 1% bromogeramine solution, taking-up is with tincture of iodine alcohol disinfecting spleen surface, remove spleen fat and coating with Dissecting scissors and tweezers, with the balanced salt solution washing once, again spleen is cut into segment and moves in the plate, with eye scissors spleen is cut into 1mm at last ³Fritter is again with balanced salt solution washing 2～3 times, to remove hemocyte, coloring matter and to shred the cell of physical abuse in the process;

B. digest and dispersion tissue's piece

The balanced salt solution that the last step was cleaned sops up, the tissue block that shreds is moved in the flask of triangle burning angle, and 5 times of amounts of pressing the tissue block volume add 0.25% trypsin solutions (PH7.6～7.8), put in 37 ℃ of water-baths to digest about 20～40 minutes.Shook one time Erlenmeyer flask every 10 minutes, so that tissue block is scattered, in order to continuing digestion, up to tissue become loose, surperficial when scared till, at this moment from water-bath, take out the Erlenmeyer flask suction and remove trypsin solution, with balanced salt solution washing 2～3 times, wash once again with 0.5% lactoalbumin hydrolysate-Hanks liquid.Add a little 0.5% hydrolysis breast egg-Hanks liquid,, filter, count with four layers of gauze with the big slightly tubule piping and druming several of bore.

C. cell counting: adopt the blood counting chamber that indicates the 0.1mm printed words to count, method of counting is identical with white blood cell count(WBC).

D. the packing of cell suspension and cultivation

By the cell counting result, cell suspension is adjusted to 500,000/ml cells suspension with nutritive medium.With cell suspension branch pack into Tissue Culture Flask (100ml) and cell rolling bottle, dividing loading amount is 1/10 of this bottle capacity, closes the lid, and carries out sign, Tissue Culture Flask and rolling bottle is placed on respectively on CO2gas incubator and the Rotary Machine cultivates then.

E. observe

Place 37 ℃ of cultured cells, need observe day by day, the main observation:

(1) whether culture pollutes, and becomes yellow and muddiness as nutrient solution, represents that this bottle pollutes.

(2) observation of cell morphological characteristic and growth phase:

Treat that cell cultures after 24 hours, can observe.If cell is grown, then want the morphological specificity of observation of cell and judge its residing growth phase (free phase, adsorption cycle, nursery stage, the phase of keeping and paracme), until forming the lymphocyte individual layer.

2. to the cultivation of going down to posterity of lymphocyte individual layer;

Before doing the passage cell cultivation, at first culturing bottle is placed microscopically to observe, to determine that cell has grown up to individual layer, can carry out the cultivation of going down to posterity of cell.

3. after the lymph passage cell grows up to individual layer, use the concanavalin A inducing culture.

After spleen lymph passage cell grew up to individual layer, replacing was kept liquid and is added the concanavalin A inducing culture simultaneously.

Inducing culture was tested to product after 24 hours:

Cell culture taken a sample after ultrasonication detect the concentration of transfer factor and antibacterial peptide.

The mensuration of transfer factor concentration: quantitatively many quantitative to transfer factor:, be a transfer factor unit with content of peptides 1mg with Folin-phenol method or spectrophotometry content of peptides with content of peptides.The transfer factor content of this explained hereafter is 4～5 times of conventional production method.

The antibacterial peptide concentration determination: adopting the Xylene Brilliant Cyanine G method to measure, is standardized solution with the bovine serum albumin, and the concentration of the antibacterial peptide of being produced is 10 times of conventional extracted amount.

The antibacterial peptide bacteriostatic experiment:

A large amount of bacteriostatic experiments shows that the antibacterial peptide micromolar concentration of being produced can be killed Gram-positive, negative bacteria and the fungi more than 85%.Its has a broad antifungal spectrum, can suppress the nearly 500 strain strain isolateds of kind surplus intestinal bacteria, staphylococcus, aerogenesis Zymomonas mobilis, suis, pasteurellosis bacillus, actinomycetes, Salmonellas, Pseudomonas aeruginosa, the mycobacterium etc. ten, and in the experiment antibacterial circle diameter on the bacterium more than the 15mm more than 85%.

The check of work in-process and finished product:

The work in-process calibrating: work in-process carry out a detection such as bacterial endotoxin, sterility test, pH value.

Finished product calibrating: finished product is made the telling test, outward appearance detections, pH value, content of peptides, SAE, proteins react, sterility test, bacterial endotoxin, undue toxicity of transfer factor and antibacterial peptide etc. respectively and is checked.

Embodiment 2:

1. be raw material with the animal peripheral blood, prepare the lymphocyte individual layer according to a conventional method.

Concrete grammar is: aseptic vein is taked 20 milliliters of animal's whole bloods, add 1% heparin solution 0.2ml anti-freezing, leave standstill about 60 minutes, with all blood plasma more than the band glueballs suction pipe absorption red corpuscle layer, the leukocytic cream that particularly is right after on the red corpuscle layer will be drawn as far as possible, be sub-packed in the sterilization centrifuge tube, with balanced salt solution repetitive scrubbing 2～3 times, 800～1200 rev/mins of centrifugal speeds, carry out the white corpuscle cultivation with containing 40 milliliters of 30% calf serum lactoalbumin hydrolysates then, being sub-packed in 100 milliliters of capacity behind the uniform mixing cultivates in square vase or the brick bottle, the jam-pack bottle stopper was put in 37 ℃ of thermostat containers or in the brick bottle machine and is cultivated, through 2～3 days, white corpuscle is evenly adherent, promptly makes the lymphocyte individual layer.

2. to the cultivation of going down to posterity of lymphocyte individual layer;

Before doing the passage cell cultivation, at first culturing bottle is placed microscopically to observe, to determine that cell has grown up to individual layer, can carry out the cultivation of going down to posterity of cell.

3. after the lymph passage cell grows up to individual layer, use the concanavalin A inducing culture.

After spleen lymph passage cell grew up to individual layer, replacing was kept liquid and is added the concanavalin A inducing culture simultaneously.

The Interventions Requested of the mensuration of transfer factor concentration, antibacterial peptide bacteriostatic experiment and result, work in-process and finished product are all with embodiment 1.

Claims (1)

1. a vitro culture induction of lymphocyte prepares the method for antibacterial peptide and transfer factor, it is characterized in that comprising the steps:

A. prepare the lymphocyte individual layer with spleen or peripheral blood;

B. to the cultivation of going down to posterity of lymphocyte individual layer;

C. after the lymph passage cell grows up to individual layer, use the concanavalin A inducing culture.