CN101684141A - Preparation method of specific transfer factor for resisting chicken bursa virus and antimicrobial peptide - Google Patents

Abstract

The invention discloses a preparation method of specific transfer factor for resisting chicken bursa virus and antimicrobial peptide, which includes the following steps: (1) immunizing experiment pigwith chicken bursa virus vaccine; (2) closely monitoring chicken bursa virus vaccine immune antibody level of experiment pig in vivo, when the antibody reaches a peak value, sterilizedly fetching splenic organ or anticoagulated blood of experiment pig, preparing lymphocyte mono-layer with splenic organ or anticoagulated blood peripheral blood; (3) executing subculture for lymphocyte mono-layer; (4) inducing and culturing the lymph subculture cell after which grows into a mono-layer with phaescolosaxin; specific transfer factor for resisting chicken bursa virus and antimicrobial peptide are obtained. The product prepared by the method does not need to be purified further, and can be treated by ultrasonic and freeze thawing simple process into a mixer for directly being used for bird in vivo, thereby playing roles for resisting avian influenza virus and bacteria, simultaneously improving immunity of bird.

Description

The preparation method of specific transfer factor for resisting chicken bursa virus and antibacterial peptide

Technical field

The present invention relates to the production method of specific transfer factor for resisting chicken bursa virus and antibacterial peptide.

Background technology

Transfer factor is a kind of lymphokine that extracts from the intact animal white corpuscle of exotic antigen sensitization, or a kind of lymphokine that extracts the animal hemocyte from the decubation after certain antigenic stimulation.The former claims non-specific transfer factor, and the latter then claims specific transfer factor.But these two kinds of transfer factor the most important thing is the transfer cell immunologic function in vivo and in vitro, as a kind of immunological reagent of adoptive immunotherapy, and the multiple vital role of performance in immunity system.

The present preparation method of transfer factor is: fresh or refrigerated spleen and lymphoglandula are removed fat and reticular tissue about 25 ℃ with animal, clean 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 the work in-process filtration sterilization 24-48 hour; The configuration of finished product and check.

Antibacterial peptide is through inducing, by the defensive peptide class active substance that resists exogenous pathogenic agent that the animal immune system of defense produces, molecular weight is little, activity is strong, has 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 new freshly-slaughtered poultry 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 conditions 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 environment.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.

The main drawback of prior art for preparing transfer factor and antibacterial peptide is not have specificity at disease, the result of treatment instability.

Summary of the invention

The objective of the invention is to overcome the antibacterial peptide and the transfer factor that prepare in the prior art and do not have specificity at disease, shortcomings such as result of treatment instability provide a kind of at treatment chicken bursal disease viral disease, the specific transfer factor for resisting chicken bursa virus of better effects and if the preparation method of antibacterial peptide.

Technical solution of the present invention is summarized as follows:

The preparation method of a kind of specific transfer factor for resisting chicken bursa virus and antibacterial peptide, be made up of following steps:

(1) with bursal disease virus of chickens vaccine 0.5-3mL intramuscular injection immunity 30-180 age in days experiment pig, 7-15 days of first immunisation are again with bursal disease virus of chickens vaccine 2mL intramuscular injection booster immunization;

(2) chicken bursa virus vaccine immune antibody horizontal in the close monitoring experiment pig body of difference, when the antibody horizontal value of peaking, aseptic experiment pig spleen or the anticoagulation taked prepares the lymphocyte individual layer with spleen or anticoagulation peripheral blood;

(3) to the cultivation of going down to posterity of lymphocyte individual layer;

(4) after the lymph passage cell grows up to individual layer, use the phytohaemagglutinin inducing culture, promptly obtain specific transfer factor for resisting chicken bursa virus and antibacterial peptide.

Advantage of the present invention:

The present invention is to use bursal disease virus of chickens immunization experiment pig earlier, with experiment pig spleen or peripheral blood are raw material again, make the lymphocyte individual layer, in vitro culture, the induction of lymphocyte individual layer can be produced the mixture of a large amount of specific transfer factor for resisting chicken bursa virus and antibacterial peptide, the specific transfer factor for resisting chicken bursa virus and the antibacterial peptide of method preparation of the present invention need not further purification, can directly mixture be used in the bird body after involving the freeze thawing simple process through ultrasonic, can play resisting chicken bursa virus and antimicrobial effect, improve the immunizing power of bird simultaneously.

Embodiment

The present invention is further illustrated below in conjunction with specific embodiment.

Embodiment 1

The preparation method of a kind of specific transfer factor for resisting chicken bursa virus and antibacterial peptide, step:

(1). immunity:

With bursal disease virus of chickens vaccine 1.0mL intramuscular injection immunity 50 age in days experiment pig, set up the blank group synchronously, after the first immunisation the 15th day, again with bursal disease virus of chickens vaccine 2mL intramuscular injection as booster immunization;

(2) close monitoring experiment pig body intradermal vaccine immune antibody level, when the antibody horizontal value of peaking, the aseptic experiment pig spleen of taking prepares the lymphocyte individual layer with spleen, and step is:

A, processing pig spleen

Get the fresh pig spleen and place aseptic Glass Containers, adding PBS (pH is 7.2) solution soaking half an hour or being dipped in mass percentage concentration is in 2% the bromogeramine solution, taking-up is with alcohol disinfecting spleen surface, remove spleen fat and coating with Dissecting scissors and tweezers, wash once with PBS (pH is 7.2) liquid, spleen being cut into segment moves in the plate again, with eye scissors spleen is cut into the 1mm3 fritter at last, use PBS (pH is 7.2) liquid washing again 3 times, to remove hemocyte, coloring matter and to shred the cell of physical abuse in the process;

B, digestion and dispersion tissue's piece

PBS (pH7.2) liquid that the last step was cleaned sops up, the tissue block that shreds is moved in the Erlenmeyer flask, adds 0.25% trypsin solutions (pH is 7.7) by 5 times of amounts of tissue block volume, puts in 37 ℃ of water-baths to digest 30 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 Erlenmeyer flask, inhale and remove trypsin solution, use PBS (pH is 7.2) liquid washing 3 times again, use 0.5% lactoalbumin hydrolysate---Hanks liquid is washed, and with the big slightly tubule piping and druming several of bore, filters with four layers of gauze;

C, cell counting

Employing indicates the blood counting chamber of 0.1mm printed words and counts, and method of counting is identical with white blood cell count(WBC);

The packing of D, cell suspension and cultivation

By the cell counting result, cell suspension is adjusted to 600,000/ml cells suspension with the DMEM nutritive medium.With cell suspension branch pack into Tissue Culture Flask and cell rolling bottle, dividing loading amount is 1/5 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, observation

Place 37 ℃ of cultured cells, need observe day by day.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.

(3) to the cultivation of going down to posterity of lymphocyte individual layer

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

(4) after the lymph passage cell grows up to individual layer, use the phytohaemagglutinin inducing culture.

After spleen lymph passage cell grew up to individual layer, replacing was kept liquid and is added the phytohaemagglutinin 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 be conventional produce 4～5 times.

The antibacterial peptide concentration determination: adopting the Xylene Brilliant Cyanine G method to measure, is standardized solution with the bovine serum albumin, and Coomassie brilliant blue G250 is a developer, the concentration of the antibacterial peptide of producing at spectrophotometric determination.The concentration of the antibacterial peptide that present method is produced is 10 times of conventional extracted amount.

Application example

200 15 age in days fryer (aseptic through physical examination) are divided into totally 5 groups of A, B, C, D, E, and 40 every group, B, C, D, E group are attacked the poison back through the strong poison of artificial challenge's 0.6ml bursal disease virus of chickens and tested.A group is healthy group, infective virus not, not administration; B organizes negative control group, infective virus, not administration; C group and D group are the product of method preparation of the present invention, and the C group is low dose group, press 0.001g/ chicken/sky injection; The D group is the pharmaceutical composition high dose group, presses 0.002g/ chicken/sky injection; The E group is pressed 0.1g/ chicken/sky administration, with medicinal day for tradition " SHUANGHUANGLIAN KOUFUYE " treatment group.The result shows, adopts the product of preparation method's preparation of specific transfer factor for resisting chicken bursa virus of the present invention and antibacterial peptide that bursal disease virus of chickens is had tangible immunization.

Experimental result is as follows:

Numbering	Group	Mortality ratio %	Curative ratio %	Efficient %
					??A	Healthy group	??0	??92.5(37/40)	??100.0(40/40)
??B	Negative control group	??67.5(27/40)	??2.5(1/40)	??32.5(13/40)
					??C	The low dose therapy group	??25.0(10/40)	??40.0(16/40)	??75.0(30/40)
??D	The high-dose therapy group	??7.5(3/40)	??65.0(26/40)	??92.5(37/40)
					??E	" SHUANGHUANGLIAN KOUFUYE " treatment group	??27.5(11/40)	??47.5(19/40)	??72.5(29/40)

The result shows, low dose therapy group, high-dose therapy group and negative control group difference highly significant (P＜0.01) illustrate that the product of preparation method's preparation of a kind of specific transfer factor for resisting chicken bursa virus of the present invention and antibacterial peptide has significant therapeutic action to bursal disease virus of chickens; Particularly the high-dose therapy group is used and obviously is better than tradition " SHUANGHUANGLIAN KOUFUYE " treatment group curative effect (P＜0.01).

The antibacterial peptide bacteriostatic experiment:

Bacteriostatic experiment shows that the antibacterial peptide micromolar concentration that the present invention produced can be killed gram-positive, gram-negative bacteria and the fungi more than 85%.Its has a broad antifungal spectrum, can suppress the nearly 150 strain strain isolateds of kind surplus staphylococcus, Hemolytic streptococcus, intestinal bacteria, aerogenesis Zymomonas mobilis, Pseudomonas aeruginosa, pasteurellosis bacillus, Salmonellas, actinomycetes, the mycobacterium etc. ten, and in the experiment antibacterial circle diameter on the bacterium more than the 22mm more than 75%.

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:

The preparation method of a kind of specific transfer factor for resisting chicken bursa virus and antibacterial peptide, step:

(1) immunity:

With bursal disease virus of chickens vaccine 1.2mL intramuscular injection immunity 30 age in days experiment pig, set up the blank group synchronously, after the first immunisation the 7th day, again with bursal disease virus of chickens vaccine 2mL intramuscular injection as second immunisation; Second immunisation the 21st day, again with avian influenza virus vaccines, bursal disease virus of chickens vaccine, avian influenza virus vaccines 2mL intramuscular injection as three immunity;

(2) close monitoring experiment pig body intradermal vaccine immune antibody level, when the antibody horizontal value of peaking, the aseptic anticoagulation of taking prepares the lymphocyte individual layer with peripheral blood, and step is:

Aseptic vein is taked 250 milliliters of animal's whole bloods, add 1.2% heparin solution 3ml anti-freezing, left standstill 70 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 PBS (pH is 7.2) liquid repetitive scrubbing 3 times, 1000 rev/mins of centrifugal speeds, carry out white corpuscle and cultivate 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 3 days, white corpuscle is evenly adherent, promptly makes the lymphocyte individual layer.

(3) 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.

(4) after the lymph passage cell grows up to individual layer, use the phytohaemagglutinin inducing culture.

After the hemolymph passage cell grew up to individual layer, replacing was kept liquid and is added the phytohaemagglutinin inducing culture simultaneously.

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

Embodiment 3

The preparation method of a kind of specific transfer factor for resisting chicken bursa virus and antibacterial peptide, be made up of following steps:

(1) with bursal disease virus of chickens vaccine 0.5mL intramuscular injection immunity 180 age in days experiment pig, first immunisation the 8th day is again with bursal disease virus of chickens vaccine 2mL intramuscular injection booster immunization;

(2) close monitoring experiment pig body intradermal vaccine immune antibody level, when the antibody horizontal value of peaking, the aseptic experiment pig spleen of taking prepares the lymphocyte individual layer with spleen;

(3) to the cultivation of going down to posterity of lymphocyte individual layer;

(4) after the lymph passage cell grows up to individual layer, use the phytohaemagglutinin inducing culture, promptly obtain specific transfer factor for resisting chicken bursa virus and antibacterial peptide.

Embodiment 4

The preparation method of a kind of specific transfer factor for resisting chicken bursa virus and antibacterial peptide, be made up of following steps:

(1) with bursal disease virus of chickens vaccine 3mL intramuscular injection immunity 100 age in days experiment pig, first immunisation the 10th day is again with bursal disease virus of chickens vaccine 2mL intramuscular injection booster immunization;

(2) close monitoring experiment pig body intradermal vaccine immune antibody level, when the antibody horizontal value of peaking, the aseptic experiment pig anticoagulation of taking prepares the lymphocyte individual layer with anticoagulation;

(3) to the cultivation of going down to posterity of lymphocyte individual layer;

(4) after the lymph passage cell grows up to individual layer, use the phytohaemagglutinin inducing culture, promptly obtain specific transfer factor for resisting chicken bursa virus and antibacterial peptide.

Claims (1)

1. the preparation method of specific transfer factor for resisting chicken bursa virus and antibacterial peptide is characterized in that being made up of following steps:

(1) with bursal disease virus of chickens vaccine 0.5-3mL intramuscular injection immunity 30-180 age in days experiment pig, 7-15 days of first immunisation are again with bursal disease virus of chickens vaccine 2mL intramuscular injection booster immunization;

(2) chicken bursa virus vaccine immune antibody horizontal in the close monitoring experiment pig body of difference, when the antibody horizontal value of peaking, aseptic experiment pig spleen or the anticoagulation taked prepares the lymphocyte individual layer with spleen or anticoagulation peripheral blood;

(3) to the cultivation of going down to posterity of lymphocyte individual layer;

(4) after the lymph passage cell grows up to individual layer, use the phytohaemagglutinin inducing culture, promptly obtain specific transfer factor for resisting chicken bursa virus and antibacterial peptide.