CN102776222B - Preparation and application of fused oral interferon for fowl - Google Patents

Abstract

Preparation and application of a fused oral interferon expression frond for fowl are disclosed. On the basis of the conserved sequences of fowl interferon alpha and gamma genes, the interferon alpha gene having great antiviral effect and the interferon gamma gene capable of improving the immunity of the organism and enhancing the constitution of the organism are fused for expression, so that the expressed alpha+gamma fused interferon has the synergetic functions of the two. In the mean time, a dunaliella bioreactor is used as an expression system; apart from that the system is capable of expressing exogenous genes, own advantages such as rich nutrition, nontoxicity, harmlessness and the like of dunaliella are also utilized; consequently, a gene algae plant for multiple purposes is obtained; the fused oral interferon expression frond for fowl prepared from the genetically modified algae plant can be applied to the fields such as prevention or treatment of fowl viroses, fowl breeding, related feed processing industry and the like; and the fused oral interferon for fowl is good in effect, green and environment-friendly, and also low in cost.

Description

A kind of fowl is expressed preparation and the application thereof of frond with pattern of fusion oraferon

Technical field

The present invention relates to field of biological pharmacy, specifically a kind of fowl is expressed its application of preparation of frond with pattern of fusion oraferon.

Background technology

At present, maximum to the harm of China poultry cultivation industry, also a class disease the most rambunctious is communicable virus disease.Be directed to such disease of bird, never have effective methods for the treatment of and means, wherein vaccine inoculation is currently to prevent that avian viral diseases from breaking out and popular major measure and key link.But along with vaccine prevention failure or inoculation not in time, the often outburst of some virus diseases in poultry cultivation, infectivity and mortality ratio are high, some can reach 100%.Once wild virus disease, the biotechnological formulations such as Interferon, rabbit are the choice drugs for the treatment of in the market.But the Interferon, rabbit of Vehicles Collected from Market application be take unit price type as main, and belongs to injection-type more, and it uses complex operation, cost is high, time-consuming, and effect is not ideal.And the production of current Avian Interferon mainly be take prokaryotic expression system as main, also utilizes eukaryotic cell expression system.Prokaryotic expression system exists and can not carry out transcribing and posttranslational modification function of protein, and mostly expression product be inclusion body in insoluble born of the same parents, yields poorly, and needs the complicated processes such as processing purifying; Eukaryotic cell expression system exists that cost costliness, conditional request are harsh, protein is vulnerable to pollute and purification difficult and be difficult to the shortcomings such as mass-producing, has all limited the application of the two system.Therefore, find the main R&D direction that the new compound Interferon, rabbit of bio-reactor production multivalence becomes current Interferon, rabbit.

Salt algae (Dunaliella salina) is a kind of unicellular Eukaryotic Algae of living in salt water, himself have advantages of that many other reactors are incomparable, as: 1) salt algae itself is nutritious, be rich in the compositions such as β-carotene, VITAMIN and protein, there is high nutritive value and medical value; 2) Halophila is biological in photosynthetic autotrophs, can directly adopt open large scale culturing, cultivates cost cheap, and productive expense is low; 3) salt algae itself is nontoxic, is a kind of natural healthcare products, can improve immunity of organisms and resistibility; 4) salt algae is a kind of natural protoplastis, it is carried out to genetic transformation simple to operate; 5) cultivation of salt algae is safe and reliable, can be to environment.6) Halophila, in eukaryote, has the working ability to albumen after transcribing and translating, and can produce nearly natural highly active protein.

Summary of the invention

In view of the production of Interferon, rabbit and application present situation for current fowl, the invention provides a kind of fowl and with pattern of fusion oraferon, express the preparation method of frond, prepared fowl is expressed frond with pattern of fusion oraferon can antiviral therapy disease, improve body immunizing power, improve bird body meat quality.Succeeding in developing of this product is all of great practical significance to the prevention of avian viral diseases and treatment, has huge economic benefit and commercial value.

Fowl is expressed preparation and the application thereof of frond with pattern of fusion oraferon, its concrete steps are as follows:

The preparation of step 1, fowl α, gamma interferon genes: the α of the listed chicken of global common sequence database GenBank, duck and three kinds of animals of goose, gamma interferon genes are carried out respectively to sequence alignment, preferences in conjunction with Dunaliella salina cell genome codon, the base sequence of three kinds of the two genes of animal is transformed to modification, synthetic three kinds of interferon-alpha sequence (sequence table sequence 1), IFN-γ sequences (sequence table sequence 2) that bird is general.According to gene order, design respectively Auele Specific Primer, by method amplification fowl α, the gamma interferon genes of PCR;

Step 2, conversion Dunaliella salina cell construction of eukaryotic expression vector: fowl α, gamma interferon genes fragment by pcr amplification are connected with pMD18-T cloning vector respectively, by cutting respectively α, γ gene fragment after double digestion evaluation separately and the evaluation of checking order, by T4 ligase enzyme, successively the two gene is connected on the carrier for expression of eukaryon of pBI series; By the recombinant vectors pBI-α+γ building for the conversion to Dunaliella salina cell;

Step 3, recombinant expression vector transform Dunaliella salina cell: the recombinant plasmid building is passed through to efficient granulated glass sphere conversion method, or transform Dunaliella salina cell by existing particle bombardment, electric shocking method, liposome mediated-method or ultrasonic wave importing method.Dunaliella salina cell after conversion, through selecting Screening of Media to cultivate, finally obtains positive transformant.The positive algae of picking falls to carrying out respectively enlarged culturing, used for follow-up molecular biology identification;

Step 4, screening and molecular biology identification to the strain of conversion algae: extract the total RNA of genome of transgenic alga strain, the goal gene being incorporated in salt algae genome is carried out to RT-PCR amplification evaluation, to finding out goal gene integration and expression status.Meanwhile, utilize western blot technology to expressing to such an extent that protein product carries out biochemical activity analysis;

Step 5, fowl are expressed the preparation of frond with pattern of fusion oraferon: will identify that the inoculum size of correct transgenic alga strain by 1% is inoculated in natural sea-water algal culture pond, carries out open large-scale cultivation.Cultivate about 10-14 days, when frustule concentration reaches 10 ⁶individual/during ml, by quiescent setting or centrifugal collection frond, to obtain fowl and express frond with pattern of fusion oraferon, can carry out next step step 6 or step 7 technique according to different purposes;

Step 6, the application in prevention or treatment avian viral diseases: for reaching this purposes, can be by the following technical solutions: under cold condition, adopt ultrasonic grinding, the salt frond of cracking adds in 5% the ratio that accounts for chicken drinking-water volume, now with the current, is controlled in 0.5-2h and has drunk;

Step 7, the application as a kind of functional feedstuff additive in poultry cultivation industry and feed processing industry: for reaching this purposes, can be by the following technical solutions: this transgenic alga strain directly or after concentrated, cryodrying, granulation, pulverizing or the processing such as mixing with other nutritive ingredient, is added in poultry feed ingredient.Addition is 1-7%, and adding form can be for solid forms such as pulvis, granules, also can be the semi-solid forms such as paste, pill, can be also liquid form.

Beneficial effect

1, the divalence Interferon, rabbit that fowl of the present invention is pattern of fusion with pattern of fusion oraferon, is that interferon alpha, the gamma gene sequences that utilizes bird conservative property stronger is basis, and α gene has strong antivirus action, can play the object for the treatment of virus disease; γ gene can regulate the effect of body physique, enhancing body immunizing power, and the two gene is carried out to amalgamation and expression α+γ, can play the synergy of said two devices effect, and therapeutic action is better.

2, the present invention has overcome the deficiency that current Interferon, rabbit production system exists, and has utilized and has collected multiple advantage for Dunaliella salina cell is all over the body as expression system.Interferon alpha+γ gene transformation Dunaliella salina cell is prepared to transgenic alga strain.This transgenic alga strain does not need the complicated course of processing such as induction, separation and purification, can be directly as the active interferon formulation poultry of throwing something and feeding, operating process is easy, time saving and energy saving.Simultaneously, the advantage of salt algae self is also utilized, and can play the effect of " strain is multiplex ", can play antiviral, improve outside immunity of organisms, can also accelerate body the speed of growth, improve the advantages such as meat quality, be a kind of nutritious green anti-virus formulation.

3, the prepared fowl of the present invention is expressed frond with pattern of fusion oraferon and can adopt natural open industrialized culture, and not only output is high, with low cost for it, and is not subject to the pollution of external microbe, and biological safety is good.

4, the ingenious close ties that exist between salt algae, poultry, Interferon, rabbit three of having utilized of the present invention: Interferon, rabbit has stronger antivirus action, virus can infect poultry, poultry can be usingd again salt algae as feed ingredient, and salt algae is a desirable green expression system.For this reason, the engineering algae strain of preparing by transgenosis means just can directly be thrown something and fed by oral way without purification processing, convenient and swift, has avoided the disadvantageous effect of the modes such as injection.The active interferon formulation of this natural green is directly undertaken by Oral administration, there is not yet report at home and abroad.

5, the fowl that prepared by the present invention is expressed frond with pattern of fusion oraferon and can not only be applied as antivirus medicine for birds, and can be used as a kind of multi-functional fodder additives and applied in poultry cultivation industry and feed processing industry, and this additive have secondary residual without any poison, without advantages such as resistance, environmental protections.

Accompanying drawing explanation

Fig. 1 is that the pcr amplification product sepharose of fowl α gene detects electrophorogram;

In figure: left side is molecular weight 2000 Marker, right side is PCR product sample

Fig. 2 is that the pcr amplification product sepharose of fowl γ gene detects electrophorogram;

In figure: M is molecular weight 2000 Marker; B is blank; P is the PCR product sample of two repetitions;

Fig. 3 is that Xba I and the BamH I double digestion sepharose of pBI-α recombinant plasmid detects electrophorogram;

In figure: left side is DNA fragmentation after pBI-α recombinant plasmid double digestion, and right side is Marker;

Fig. 4 is that BamH I and the Sac I double digestion sepharose of pBI-α+γ recombinant plasmid detects electrophorogram;

In figure: left side is DNA fragmentation after pBI-α+γ recombinant plasmid double digestion, and right side is Marker;

Fig. 5 is for transforming carrier for expression of eukaryon pBI-α+γ structural representation of Dunaliella salina cell

Fig. 6 is the solid medium screening and culturing result figure of transgenic alga strain;

In figure: the negative control group of left side plate, the growing state of wild-type Dunaliella salina cell, drops out existing without single algae; Right side plate is for transforming the growing state of algae strain, the growth that has as seen a plurality of single algaes to fall;

Fig. 7 is for carrying out the pcr amplification evaluation figure of α gene to positive transformant;

In figure: M is molecular weight 2000 Marker; The negative contrast of W, i.e. the salt algae strain of wild-type; T1, T2 are respectively the positive transformant of two strains;

Fig. 8 is for to carry out Western blot analytical results figure to expression product;

In figure: left side is albumen Marker, the interferon protein band of right side for merging

Embodiment

The method providing in < < fine works molecular biology experiment guide (the 4th edition) the > > that the present invention's genetically engineered elementary operation technology used is published by scientific publication for 2005 is carried out, or operates according to the working instructions of bought reagent, product.

The embodiment of the present invention relates to following material:

The carrier for expression of eukaryon of pMD18-T cloning vector, pBI series is bought the precious biological company limited in Dalian; Intestinal bacteria competence is bought the precious biological company limited in Dalian; Restriction enzyme Xba I, BamH I, Sac I are purchased from the precious biological company limited in Dalian; T4 DNA ligase is purchased from the precious biological company limited in Dalian; Trizol reagent is bought the company in American I nvitrogen; PCR cleaning agents box and glue reclaim test kit purchased from U.S. Axygene company; The synthetic of Auele Specific Primer synthesized by the Shanghai biological company limited of raw work;

Fowl is expressed preparation and the application thereof of frond with pattern of fusion oraferon, its concrete steps are as follows:

The amplification of step 1, fowl α, gamma interferon genes

1) preparation of fowl alpha-IFN gene

To the listed chicken of global common sequence database GenBank, (accession number is respectively DQ226092 to the alpha-IFN gene of duck and goose, EF053034 and AY524422) carry out sequence alignment, in conjunction with the genomic codon-bias of salt algae expression system, the gene order of three kinds of animals is transformed to modification, the higher amino acid of the frequency of occurrences is assigned to corresponding position, avoid the appearance stopping and non-coding base combines, synthetic the promotor of three kinds of general for animal be ATG, terminator is the interferon-alpha sequence (sequence table sequence 1) of TAA, according to this sequences Design, go out pair of primers, upstream primer: 5 ' tCTAGAaTGGCTGGGCCATCAGCCCC3 ', downstream primer: 5 ' gGATCCcGTGTTGGTGCGGGT 3 ', underscore indication is Xba I and the BamH I restriction enzyme site that upstream and downstream primer is introduced respectively.Then adopt following response procedures to carry out pcr amplification: 94 ℃ of denaturation 5min, 30 circulations (72 ℃ are extended 70S for 94 ℃ of sex change 40S, 53 ℃ of annealing 40S), termination reaction after 72 ℃ of extension 10min.PCR product identifies through 1% agarose gel electrophoresis, and result demonstrates the gene fragment (result as shown in Figure 1) of big or small about 600bp;

2) preparation of fowl gamma interferon genes

Gamma interferon genes (accession number is respectively AY163160, AY166850 and AY524421) to chicken, duck and the goose of the login of global common sequence database GenBank carries out sequence alignment, in conjunction with the genomic codon-bias of salt algae expression system, the higher amino acid of the frequency of occurrences is assigned to corresponding position, synthetic the promotor of three kinds of general for animal be ATG, terminator is the IFN-γ sequence (sequence table sequence 2) of TAA, according to this sequences Design, go out pair of primers, upstream primer: 5 ' gGATCCaCTTGCACTACCCAGACTTAC3 ', downstream primer: 5 ' gAGCTCtTACGTGTTGGTGCGGGTGA 3 ', underscore indication is BamH I and the Sac I restriction enzyme site that upstream and downstream primer is introduced respectively.Then adopt following condition to carry out pcr amplification: 94 ℃ of pre-4 min of change, 28 circulations (72 ℃ are extended 100S for 94 ℃ of sex change 45 S, 56 ℃ of annealing 50S), termination reaction after 72 ℃ of extension 10min.PCR product identifies through 1% agarose gel electrophoresis, and result demonstrates the gene fragment (result as shown in Figure 2) of big or small about 500bp.

The clone of step 2, PCR product and evaluation

1) clone of interferon alpha, γ gene fragment

Before PCR product connects pMD18-T cloning vector, the purifying of advanced performing PCR product reclaims.Adopt PCR cleaning agents box to reclaim respectively α, γ gene fragment, according to appended specification sheets, operate.Recovery is obtained to α fragment gene fragment adopts restriction enzyme XbaI and BamHI to carry out double digestion, γ gene fragment adopts restriction enzyme BamH I and Sac I to carry out double digestion, then connect with the pMD18-T cloning vector through identical double digestion respectively, linked system is as follows: 2 10 times of μ l T4 DNA ligase damping fluids, 1 μ l pMD18-T carrier, 7 μ l α or γ gene fragment, 1 μ l T4 DNA ligase enzyme, supplement ddH2O to cumulative volume 20 μ l, then ligation system is placed and is spent the night in 16 ℃ of water-baths.

2) enzyme of restructuring pMD18-T-α (or γ) the cloning vector evaluation of cutting and check order

Adopt CaCl ₂heat shock method is converted into intestinal bacteria competence by cloning vector pMD18-T-α (or γ), adopts blue hickie screening, 37 ℃ of incubated overnight.Then picking positive colony, after enlarged culturing, adopt alkaline lysis to extract in a small amount plasmid, plasmid is carried out respectively to double digestion evaluation, and (recombinant plasmid pMD18-T-α adopts restriction enzyme XbaI and BamH I to carry out double digestion, recombinant plasmid pMD18-T-γ is used restriction enzyme BamH I and Sac I to carry out double digestion), can detection cut out the gene fragment of expection.Then choosing enzyme cuts and identifies that correct positive colony sends the evaluation of checking order of the Shanghai biological company limited of raw work.

The structure of step 3, conversion Dunaliella salina cell pBI-α+γ carrier for expression of eukaryon

Enzyme is cut and checked order and identify and to carry out respectively double digestion by all correct recombinant vectors pMD18-T-α (or γ) (recombinant plasmid pMD18-T-α adopts restriction enzyme Xba I and BamH I to carry out double digestion, recombinant plasmid pMD18-T-γ restriction enzyme BamH I and Sac I carry out double digestion), then adopt Axygene company glue to reclaim test kit and reclaim interferon alpha, γ gene fragment, according to its appended specification sheets, operate; First the carrier for expression of eukaryon of pBI series is carried out to restriction enzyme Xba I and BamH I double digestion, the enzyme system of cutting is: 1.5 μ l Xba I enzymes, 1.5 μ l BamH I enzymes, the expression vector of 7 μ lpBI series eucaryons, 2 μ l buffer, benefit adds water to cumulative volume 20 μ l, in 37 ℃ of water-bath enzymes, cuts and spends the night, and then by glue, reclaims test kit and reclaims carrier segments.The alpha gene fragment reclaiming is connected with carrier segments.Linked system is: 2 μ l carrier segments, and 10 μ l alpha gene fragments, 2 10 times of μ l T4 DNA ligase damping fluids, T4 DNA ligase 1.5 μ l, mend and add water to cumulative volume 20 μ l, then in 16 ℃ of water-baths, connect and spend the night;

Adopt CaCl next day ₂heat shock method is transformed into intestinal bacteria competence by this linked system respectively, adopts blue hickie screening, and the positive bacterium colony of picking carries out the extraction of plasmid after cultivating.Extract plasmid and adopt restriction enzyme XbaI and BamH I to carry out double digestion evaluation, enzyme cuts system and condition is the same, and result as shown in Figure 3, has cut out the fragment of about 600bp.Identify that correct recombinant plasmid carries out BamH I and Sac I double digestion, the enzyme system of cutting is: 1.5 μ l Sac I enzymes, 1.5 μ l BamH I enzymes, 7 μ l pBI-α carriers, 2 10 times of μ l restriction enzyme buffer, benefit adds water to cumulative volume 20 μ l, in 37 ℃ of water-bath enzymes, cuts and spends the night, and then by glue, reclaims test kit and reclaims carrier segments.The carrier segments reclaiming is connected with γ gene fragment, linked system and condition are with the connection of alpha gene fragment, then recombinant plasmid is carried out to restriction enzyme BamH I and Sac I double digestion evaluation (shown in Fig. 4), finally successfully build pBI-α+γ carrier for expression of eukaryon, the structure of this carrier as shown in Figure 5.

The conversion of step 4, Dunaliella salina cell and the screening and culturing of transformant

Adopt granulated glass sphere conversion method that recombinant plasmid pBI-α+γ is transformed to frustule, main operational steps is as follows: collect the Dunaliella salina cell 800 μ l of logarithmic phase, cell concn is 10 ⁶individual cell/ml, then adds 150 μ l 20% polyoxyethylene glycol and 90 μ l recombinant plasmids (concentration is 0.55 μ g/ μ l), under the condition existing, in rotating speed 2400 rpm vortex 12 s, completes conversion process at 300 mg granulated glass spherees.After the complete sedimentation of granulated glass sphere, transformation system is secretly cultivated to 12h with PKS substratum, then 3 days (light: dark than being 12:12) is cultivated in normal illumination.Adopt the centrifugal 5min of 2000rpm to collect Dunaliella salina cell, select substratum to carry out screening and culturing cell applying solid, until the appearance (as shown in Figure 6) that visible single algae falls.Meanwhile, be provided with negative control group in conversion process, do not add the conversion operation group of recombinant plasmid, the experimental implementation process of this group is identical.

Step 5, express alpha+γ fused interferon transform the molecular biology identification of algae strain

First from the solid culture plate of screening, the positive single algae of picking falls, and inoculation 5ml PKS liquid nutrient medium test tube is cultivated 7 days.Then by 10% inoculum size, inoculate Erlenmeyer flask enlarged culturing, be cultured to cell concn and reach 10 ⁵individual/during ml, centrifugal collection salt frond.Utilize Trizol reagent, according to its appended specification sheets, operate, extract total RNA of salt algae after transforming, adopt the method for RT-PCR to carry out the amplification of α gene, to find out integration and the expression of goal gene.PCR product is identified by 1% sepharose, can be accessed the gene fragment (as shown in Figure 7) that specificity is strong, size meets expection.Presentation of results goal gene has been incorporated in the genome of salt algae, and has obtained effective transcriptional expression.Meanwhile, utilize Western blot to carry out Analysis on Biological Activity to the target protein merging.Result shows, at molecular weight, is that 40KD place demonstrates special band, illustrates that α+γ fused interferon of expressing keeps its natural biological function (as shown in Figure 8).

Step 6, the transgenic alga strain application in avian viral diseases prevention and treatment as antiviral

1) the transgenic saline frond with cracking acts on chicken body by water way, carries out antiviral prevention Protection

Respectively transgenic saline algae and unconverted wild Dunaliella salina cell are carried out to centrifugal collection, under cold condition, adopt ultrasonic grinding, parameter is 400W, broken 10S interval 3S, broken 20 times altogether.The salt frond of cracking adds in 5% the ratio that accounts for chicken drinking-water volume, now with the current, is preferably controlled in 0.5-2h and has drunk.Under the condition of normally feeding, give whole day and repeatedly drink water, continuous application 3 days.Select the SPF chicken of 15 age in days health, be divided at random three groups, 20 every group, establish three groups of repetitions for every group.A group: blank group, in chicken breeding process, give normal general tap water, do not add Dunaliella salina cell; B group: negative control group, in the breeding process of chicken, contains the tap water of 5% wild-type salt frond; C group, treatment comparative group, in the breeding process of chicken, contains the tap water of 5% transgenic saline frond.Group used is on the basis of repeatedly drinking water for three days on end, and the 4th day starts, and three treated animal unifications are thrown something and fed containing infectious bursal disease material, and water way carries out as above-mentioned, drinks continuously 7 days, records every group of chicken incidence every day.

Experimental result (as shown in table 1): the sickness rate of blank group and negative control group chicken reaches 100%; the cumulative mortality of the two is respectively 56.5% and 53.5%; and the not morbidity and dead for the treatment of group chicken; show that transgenic saline algae strain is by water way effect chicken body, the Effective Vate of Protection that it is carried out to prophylaxis of viral diseases reaches 100%.

The method of calculation for the treatment of group protection ratio: protection ratio=(1-treatment group death toll/treatment group animal number) * 100%

The method of calculation of blank group mortality ratio: mortality ratio=(blank group death toll/blank treated animal sum) * 100%

The method of calculation of negative control group chicken death rate: mortality ratio=(negative control group death toll/negative control group animal number) * 100%

The strain of table 1 transgenic alga is the antiviral prevention Protection to chicken body by water way

2) directly the transgenic saline frond of live body is admixed to feed, by oral way, act on duck body, carry out antiviral result for the treatment of evaluation

Transgenic saline algae and unconverted wild salt algae are carried out respectively to centrifugal collection, and the ratio that accounts for feed 5% according to Dunaliella salina cell weight in wet base is added in duck feed and is gone, and waits to feed after fully mixing.The 5 age in days ducks that suffer from initial stage (infecting in 1 day) viral hepatitis through laboratory diagnosis are divided into three groups at random, 10 every group, establish three groups of repetitions for every group.A group: blank group, to the duck normal feed of throwing something and feeding, does not add Dunaliella salina cell; B group: negative control group, duck is thrown something and fed containing the feed of 7% wild-type salt frond; C group, treatment comparative group, throws something and feeds containing the feed of 7% transgenic saline frond to duck.The every natural gift of group used are thrown something and fed for three times, throw something and feed continuously 7 days, record every group of chicken incidence every day.

Experimental result (as shown in table 2): the cumulative mortality of blank group and negative control group reaches respectively 87% and 83%, the cumulative mortality for the treatment of group is about 20%, show that this α+γ fused interferon has stronger antiviral therapy effect, makes mortality ratio effectively reduce 65%.

The method of calculation of blank group mortality ratio: mortality ratio=(blank group death toll/blank treated animal sum) * 100%

The method of calculation of negative control group mortality ratio: mortality ratio=(negative control group death toll/negative control group animal number) * 100%

The method of calculation for the treatment of group mortality ratio: mortality ratio=(treatment group death toll/treatment group animal number) * 100%

The strain of table 2 transgenic alga is the antiviral therapy effect identification to duck body by oral way

Step 7, the transgenic alga strain application in poultry cultivation industry as fodder additives

Get transgenic saline frustule, after centrifugal collection, under cold condition, make the forms such as Powdered, particulate state and expanded shape, also can according to the ratio of 1:1, mix with sticky agent gelatin, make paste fluid form, by 7% amount of feed proportion, add the animal of throwing something and feeding after mixing; Also can join in some other fodder additives component: as VITAMIN, antioxidant, protein powder, pharmaceutical compositions etc., after mixing with it, by 2% amount that accounts for feed, add, after mixing, throw something and feed.Use the cultivated animals of this fodder additives, its growth is very fast, and body weight increases, and plumage look obviously bright-coloured, naturally glossy, and meat taste is good, delicate smooth.

Described salt algae is the common salt algae that do not make a variation in the present invention, by general Yan Zao plant or other cell biological laboratories, all can obtain;

Described intestinal bacteria competence, is Bacillus coli communis competence, by market, is bought and can be obtained;

Described pMD18-T cloning vector and the carrier for expression of eukaryon of pBI series are general carrier, can be bought and be obtained by market;

The composition of described PKS substratum: contain 1.5 mol sodium-chlor in every liter of PKS substratum, 10 m mol saltpetre, 50 m mol sodium bicarbonates, 5 m mol magnesium sulfate heptahydrates, 0.4 mmol potassium primary phosphate, 2 μ mol ferric chloride (FeCl36H2O)s, 5 μ mol ethylenediamine tetraacetic acid (EDTA)s, 7 μ mol tetra-water manganous chloride, 1 μ mol copper chloride dihydrate, 1 μ mol zinc chloride, 1 μ mol CoCL2 6H2O, 1 μ mol tetra-water ammonium molybdates, 185 μ mol boric acid, 0.2 mmol calcium chloride _,surplus is water;

Described solid is selected the composition of substratum: every liter of substratum contains 1.5 mol sodium-chlor, 10 m mol saltpetre, 50 m mol sodium bicarbonates, 5 m mol magnesium sulfate heptahydrates, 0.4 mmol potassium primary phosphate, 2 μ mol ferric chloride (FeCl36H2O)s, 5 μ mol ethylenediamine tetraacetic acid (EDTA)s, 7 μ mol tetra-water manganous chloride, 1 μ mol copper chloride dihydrate, 1 μ mol zinc chloride, 1 μ mol CoCL2 6H2O, 1 μ mol tetra-water ammonium molybdates, 185 μ mol boric acid, 0.2 mmol calcium chloride, 10g agar powder and 3mg grass fourth phosphine, surplus is water;

The composition of 20% described polyoxyethylene glycol: contain 20g polyoxyethylene glycol in every 100ml water;

SEQUENCE LISTING

<110> University Of Science and Technology Of He'nan

Preparation and the application thereof of pattern of fusion oraferon for <120> fowl

<130> 1

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 576

<212> DNA

<213> artificial sequence

<400> 1

atggctgggc catcagcccc accaccacca gccatctaca gcatcctggc gctcacgctc 60

ctcctgacgc ctctcgccaa cgccgcctcc tgctgccccc tgcgcctcca ccacagcgcc 120

ttcttctggg acagcagcca gcagctctgc aacatggctc ccagccccac acagccctgc 180

ccgcagcaac acgcgccttg ctccttcccg gacaccctcc tggacaccac tagcacgcag 240

caagccgcac acaccaccct ccacctcctc caacacctct tcgacaccct cagcagcccc 300

agcacccccg cgcactggct ccacaccgca cgccacgacc tcctcaacca gctccagcac 360

cacatccacc acctcgagcg ctgcttccca gccgacgcca cgcgcttcca caggcgaggg 420

ccccgcaacc ttcacctcag caccaacaag tacttcggct gcatcctaca cttcatccag 480

aacgacaact acagcccctg cgcatgggac cacgtccgcc tcgaggctca cgcctgcttc 540

cagcgcatcc accgcctcac ccgcaccaac acgtaa 576

<210> 2

<211> 435

<212> DNA

<213> artificial sequence

<400> 2

atgacttgca ctacccagac ttacaacttg ctcgttgtct ccgtcatcat gttttattat 60

ggacgaagtg caagtagtct aagtcaactt caacatcaag tagatatagc gaaactgaaa 120

gctaacttta gttcaagtga ttcagctgta gctgacggtg ttcctataat taaagtgaaa 180

ctgaagaact tgagagagga aaatcagatc aggatcatac tgagccagat tgtttcgttg 240

tacatgctaa agcttgcaaa catgtacaag acaaagccgc acaccaaaca catatctgag 300

gagctctata ctctgaaaaa caaccttcct gatggcgtga agaaggtgaa agtgatcctg 360

gtcagcatca aacaggtgct ggtgaatact ccgagtttca aaaggaacat gagccagtct 420

agatgcagat gttaa 435

Claims (3)

1. fowl is expressed a preparation for frond with pattern of fusion oraferon, it is characterized in that: its concrete preparation process is as follows:

1) preparation of fowl α, gamma interferon genes: design respectively two pairs of Auele Specific Primers: fowl alpha-IFN gene primer sequence is: upstream primer: 5 ' tCTAGAaTGGCTGGGCCATCAGCCCC3 ', downstream primer: 5 ' gGATCCcGTGTTGGTGCGGGT 3 ', underscore indication is Xba I and the BamH I restriction enzyme site that upstream and downstream primer is introduced respectively; Gamma interferon genes gene primer sequence is: upstream primer: 5 ' gGATCCaCTTGCACTACCCAGACTTAC3 ', downstream primer: 5 ' gAGCTCtTACGTGTTGGTGCGGGTGA 3 ', underscore indication is BamHI and the Sac I restriction enzyme site that upstream and downstream primer is introduced respectively, then the method by PCR amplifies respectively fowl alpha-IFN gene and fowl gamma interferon genes; Fowl alpha-IFN gene sequence is as shown in sequence table sequence 1, and fowl gamma interferon genes sequence is as shown in sequence table sequence 2;

2) transform Dunaliella salina cell construction of eukaryotic expression vector: fowl α, gamma interferon genes fragment by pcr amplification are connected with pMD18-T cloning vector respectively, by cutting respectively α, γ gene fragment after double digestion evaluation separately and the evaluation of checking order, by T4 ligase enzyme, successively the two gene is connected on the carrier for expression of eukaryon of pBI series; By the recombinant vectors pBI-α+γ building for the conversion to Dunaliella salina cell;

3) recombinant expression vector transforms Dunaliella salina cell: the recombinant plasmid building is passed through to granulated glass sphere conversion method, or transform Dunaliella salina cell by particle bombardment, electric shocking method, liposome mediated-method or ultrasonic wave importing method; Dunaliella salina cell after conversion selects Screening of Media to cultivate through solid, and the final positive that obtains transforms algae strain; Enlarged culturing is carried out in the positive algae strain of picking, used for follow-up molecular biology identification;

4) to transforming screening and the molecular biology identification of algae strain: the total RNA of genome that extracts transgenic alga strain, the goal gene being incorporated in salt algae genome is carried out to RT-PCR amplification evaluation, to finding out goal gene, integrate and expression status, meanwhile, utilize western blot technology to expressing to such an extent that protein product carries out biochemical activity analysis;

5) fowl is expressed the preparation of frond with pattern of fusion oraferon: by identifying that the inoculum size of correct transgenic alga strain by 1% is inoculated in natural sea-water algal culture pond, carry out open large-scale cultivation, cultivate 10-14 days, when frustule concentration reaches 10 ⁶individual/during ml, by quiescent setting or centrifugal collection frond, to obtain fowl and express frond with pattern of fusion oraferon.

2. according to the prepared fowl of claim 1, with pattern of fusion oraferon, express the application of frond in the medicine of preparing avian viral diseases prevention or treatment.

3. according to the prepared fowl of claim 1, with pattern of fusion oraferon, express frond applies in preparing poultry farming fodder additives.

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