CN102008720B - Clostridium welchii disease resistant transgenic plant vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses a clostridium welchii disease resistant transgenic plant vaccine and a preparation method thereof. At present, the protective ratio on immunization of clostridium welchii disease resistant vaccines is lower. The preparation method of the clostridium welchii disease resistant transgenic plant vaccine comprises the steps of: (1) with a plasmid or disease sample containing coded clostridium welchii toxin as a template, carrying out PCR (Polymerase Chain Reaction) or RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification to obtain a clostridium welchii toxin gene, carrying out enzyme cutting on the obtained clostridium welchii toxin gene or the Ti or Ri plasmid respectively, recycling a target gene and a plant expression vector segment, connecting with and converting escherichia coli, carrying out enzyme cutting, PCR and sequencing identification on the recombinant Ti or Ri plasmid, converting agrobacterium tume faciens or agrobacterium rhizogene with the recombinant plasmid, and screening and identifying positive recombinant agrobacterium; and (2) carrying out vernalization, germination and splicing on plant seeds to obtain explants, infecting the explants respectively with the recombinant agrobacterium in the step (1), carrying out screening, subculturing, rooting and transplanting on the infected explants to obtain transformed plants, and screening and identifying positive transgenic plants.

Description

Anti-clostridieum welchii disease Transgenic Plant Vaccines and preparation method thereof

Technical field:

The present invention relates to a kind of anti-clostridieum welchii disease Transgenic Plant Vaccines and preparation method thereof.

Background technology:

Clostridieum welchii (Clostridium welchii) is one of animal intestinal normal flora also known as bacillus perfringens (Clostridium perfringens), is also the former act of violence causing clostridieum welchii disease.Its pathogeny is under the inducements such as feeding environment and feedstuff flip-flop, clostridieum welchii amount reproduction in intestinal, the toxin produced absorbs through digestive tract, animal is caused to occur enterotoxemia, necrotic enteritis, sheep pulpy kidney disease, piglet red dysentery, lamb dysentery, rabbit hemorrhagic dysentery, gangrene after trauma, animal Sudden Death Syndrome etc.Different according to thalline secretion extracellular toxin, clostridieum welchii is divided into five serotypes, i.e. A, B, C, D, E type (see table 1).Extracellular toxin is the virulence factor of clostridieum welchii, 15 kinds more than are found that there is at present, but the lethal factor only has 4 kinds, i.e. α, β, ε and ι, wherein the most basic, most important virulence factor---alpha toxin (C.perfringens alpha toxin, CPA) is the common virulence factor of A ~ E type.Alpha toxin has phospholipase C (PhospholipaseC, PLC) and sphingomyelinase (Sphingomyelinase) active, energy hydrolyzed lecithin and sphingomyelins make membranolysis, thus have the characteristics such as hemolytic, cytotoxicity, lethal and cutaneous necrosis.

The various clostridieum welchii of table 1 and Major Secretory toxin thereof

Note: "+" expression excretes poison; "-" expression does not excrete poison.

Since the eighties has report a mid-term, the whole nation about have in the rabbit of 20 multiple provinces and cities, cattle, sheep, swinery occurred a kind of morbidity be super acute process, dead fast, the pertinacious disease that mortality rate is high, names as " Sudden Death Syndrome " according to anxious, the dead fast feature of its morbidity.The Ministry of Agriculture the mid-90 once organized scientific research personnel in Shandong, Sichuan, Henan, Jilin, Hubei, the ground such as Ningxia investigates, through proving that domestic animal " Sudden Death Syndrome " has direct relation with the infection of clostridieum welchii to Antigen isolation and identification.The sickness rate of China various places annual ruminant necrotic enteritis or enterotoxemia, lamb and piglet red dysentery is in rising trend, has carried out huge economic loss to cultivation industrial belt.Clostridieum welchii is also larger to mankind's harm, particularly during a world war, the M & M of injured soldier's gas gangrene is all very high, gas gangrene with local edema, aerogenesis, muscular death and systemic toxicity profiles for feature, in tommy atkins, this disease accounts for the ratio of wound up to 12%, causes the life that wound infection has seized 150,000 soldiers; In the Wenchuan violent earthquake of Sichuan Province of China, a lot of people, also because wound infection clostridieum welchii, produces gas gangrene and amputation of having to.Meanwhile, clostridieum welchii can also cause human foods poisoning, and in western countries such as the U.S., the alimentary toxicosis that clostridieum welchii causes comes second; At our this microbial alimentary toxicosis of country, be only second to Salmonella and staphylococcus aureus.

The pathogenic mainly extracellular toxin effect of clostridieum welchii is brought out, antibiotic can only work to clostridieum welchii itself, toxin in blood has been discharged into without any effect to antibacterial, this just causes primary disease once occur, often have little time treatment namely very fast dead, visible, the meaning of prevention clostridieum welchii disease is far longer than treatment.In order to prevent and treat the animal necrotic enteritis that clostridieum welchii causes, the method usually adopted at present is in feedstuff, add Antibiotics medicated feed additive, but this usually can bring the problem of residue of veterinary drug, and may cause the generation of Resistant strain.So vaccination is the main path of this disease of prevention, produce the focus that efficient protectiveness vaccine is research and development always.The toxoid vaccine, multiple vaccines, the recombinant vaccine that generally use at present, though there is certain effect protective rate all lower; Also there is certain defect in conventional vaccine, as deactivation is not thorough, virulence returns strong equivalent risk, and cost is higher, so urgently a kind of novel method for the treatment of occurs.

The ectotoxic chemical composition of clostridieum welchii is protein, has good immunogenicity, and foreign study shows that toxoid vaccine can reach infallible effect to the prevention of primary disease.Guo Mingzhang (1988) reports, after rabbit immunization clostridieum welchii vaccine the 5th day starts to occur toxin antibody in serum, and within about 1 month, reaching summit is 6.0log2.Liu Wenbo (2002) have developed clostridieum welchii toxoid vaccine at home first, can produce the toxin antibody of higher level after rabbit immunization.Chai Tongjie (2005) research shows that first week anti-toxin levels is 1.125log2 after clostridieum welchii toxoid aluminium glue Seedling immunizing rabbit, and reach 7.125log2 during peak, 17 weeks antibody titers are 4.7log2.Lv Jing (2009) compares A type clostridieum welchii toxoid oil emulsion vaccine and inactivated vaccine immunizing rabbit, inactivated vaccine inoculation position all has granuloma phenomenon, toxoid vaccine does not almost produce untoward reaction except having individually slight granuloma, and visible oil emulsion toxoid vaccine is high to animal safety; The rear strong virus attack of immunity, oil emulsion toxoid vaccine protective rate reaches 100%, does not occur untoward reaction, and inactivated vaccine reaches the protective rate of 75%, and not dead rabbit lassitude, there is mild diarrhea; After immunizing rabbit, antagonist level compares, and the immune effect of oil emulsion toxoid vaccine is obviously better than inactivated vaccine, studies basically identical with external Uzal.The prophylactic principle of the present invention anti-clostridieum welchii disease Transgenic Plant Vaccines is similar to toxoid vaccine.

At present formaldehyde is still to the chemical substance that extracellular toxin deactivation is conventional, its inactivating efficacy is mainly relevant with pH with the concentration of formaldehyde, temperature, action time, adopt high-concentration formaldehyde and high-temperature inactivation, although can inactivation time be reduced, but also very large to the loss of antigen, it is active that formalin has reduction energy Profilin matter, also relates to the antigenicity retaining toxin and the problem eliminating its toxicity to greatest extent, because the factor affecting deactivation is a lot, be difficult to reach a desirable deactivation condition.After the welch's fusiform bacillus alpha endotoxin toxin immunity sheep of formaldehyde detoxification; there is protective effect; but also may contain other antigenic component of clostridieum welchii in toxoid; need repeatedly lumbar injection immunity; and there is formalin-inactivated and thoroughly do not cause clostridieum welchii environmental pollution, greatest problem is that toxoid induce antibody generation required time often exceedes the time of gas gangrene symptom appearance.Simultaneously, inactivated vaccine not easily stimulates body to produce local mucosa-immune, mediating cellular immune ability is more weak, reduce the immune efficacy of inactivated vaccine, and manufacturing process is more loaded down with trivial details, having to pass through non-oral routes inoculation could induce body to produce cyclicity antibody, is difficult to produce secreting type local antibody, therefore can not stops that toxin enters body from mucosal absorption.

Summary of the invention:

The object of this invention is to provide oral anti-clostridieum welchii disease Transgenic Plant Vaccines of a kind of energy and preparation method thereof.The present invention is directed to the pathogeny of clostridieum welchii disease, namely a large amount of toxin is produced due to clostridieum welchii Fast-propagation in intestinal, the feature causing systemic toxicity reaction is absorbed through digestive tract, clostridieum welchii toxin gene is proceeded to plant by design, by Oral transgene plant vaccine, effective stimulus Intestinal Mucosal Immunity system, blocks the primary defence line of virulence factor invasion body, for science prevention clostridieum welchii disease provides new approach.

Above-mentioned object is realized by following technical scheme:

The manufacture method of anti-clostridieum welchii disease Transgenic Plant Vaccines, the method comprises:

(1) with containing the plasmid of coding clostridieum welchii alpha toxin or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain welch's fusiform bacillus alpha toxin gene, the welch's fusiform bacillus alpha toxin gene of acquisition and Ti-plasmids are carried out enzyme action respectively, reclaim genes of interest and plant expression vector fragment, connect and transformation of E. coli, enzyme action is carried out to restructuring Ti, PCR and order-checking qualification, use recombinant plasmid transformed agrobacterium tumefaciens, screening, PCR qualification obtains positive restructuring agrobacterium tumefaciens pA (Agrobacterium tumefaciens LBA 4404/pA): be preserved in China typical culture collection center on June 2nd, 2010, preserving number is: CCTCC NO:M 2010130, depositary institution address: Wuhan University of Wuhan City of Hubei China province China typical culture collection center.

(2) plant seed is through vernalization, sprouting, montage, obtain explant, explant is infected respectively with the agrobacterium tumefaciens pA of restructuring in step (1), by screening, subculture, take root, transplant, obtain transformed plant, Screening and Identification transgenic positive plant, extracts transgenic plant genome or albumen carries out Southern blot, Northern blot or Western blot identifies, proves that clostridieum welchii toxin is expressed in plant.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, the clostridieum welchii alpha toxin described in step (1) also can adopt clostridieum welchii alpha toxin and/or clostridieum welchii β toxin and/or clostridieum welchii ε toxin and/or clostridieum welchii ι toxin; Described Ti-plasmids also can adopt Ri plasmid, and the agrobacterium tumefaciens simultaneously adopts Agrobacterium rhizogenes.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, plant described in step (2) comprises herbage or vegetable or crops or fruit or model plant, described herbage comprises Herba Medicaginis, described vegetable comprises Rhizoma Solani tuber osi, described crops comprise Semen Maydis, described fruit comprises Fructus Lycopersici esculenti, and described model plant comprises Nicotiana tabacum L..

Utilize the anti-clostridieum welchii disease Transgenic Plant Vaccines that above-mentioned method makes, described anti-clostridieum welchii disease Transgenic Plant Vaccines is directly oral, is also through extracting, being processed into oral or ejection preparation use by the proteantigen of expression of plants.

A manufacture method for anti-clostridieum welchii disease Transgenic Plant Vaccines, the method comprises:

(1) with containing the plasmid of mucosal adjuvant LTB (LT-B) or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain mucosal adjuvant LT-B gene, with containing the plasmid of coding clostridieum welchii alpha toxin or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain welch's fusiform bacillus alpha toxin gene, described mucosal adjuvant LT-B gene and described welch's fusiform bacillus alpha toxin gene are merged and obtains fusion gene, acquisition fusion gene and Ti-plasmids are carried out enzyme action respectively, reclaim genes of interest and plant expression vector fragment, connect and transformation of E. coli, enzyme action is carried out to restructuring Ti-plasmids, PCR and order-checking qualification, use recombinant plasmid transformed agrobacterium tumefaciens, screening, PCR qualification obtains positive restructuring agrobacterium tumefaciens pLA (Agrobacterium tumefaciens LBA 4404/pLA): be preserved in China typical culture collection center on June 2nd, 2010, preserving number is: CCTCC NO:M 2010131, depositary institution address: Wuhan University of Wuhan City of Hubei China province China typical culture collection center.

(2) plant seed is through vernalization, sprouting, montage, obtain explant, explant is infected respectively with the agrobacterium tumefaciens pLA of restructuring in step (1), by screening, subculture, take root, transplant, obtain transformed plant, Screening and Identification transgenic positive plant, extracts transgenic plant genome or albumen carries out Southern blot, Northern blot or Western blot identifies, proves that clostridieum welchii toxin is expressed in plant.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, the LTB (LT-B) described in step (1) also can adopt b subunit of cholera toxin (CT-B).

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, the clostridieum welchii alpha toxin described in step (1) also can adopt clostridieum welchii alpha toxin and/or clostridieum welchii β toxin and/or clostridieum welchii ε toxin and/or clostridieum welchii ι toxin; Described Ti-plasmids also can adopt Ri plasmid, and the agrobacterium tumefaciens simultaneously adopts Agrobacterium rhizogenes.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, plant described in step (2) comprises herbage or vegetable or crops or fruit or model plant, described herbage comprises Herba Medicaginis, described vegetable comprises Rhizoma Solani tuber osi, described crops comprise Semen Maydis, described fruit comprises Fructus Lycopersici esculenti, and described model plant comprises Nicotiana tabacum L..

The anti-clostridieum welchii disease Transgenic Plant Vaccines utilizing said method to make, described anti-clostridieum welchii disease Transgenic Plant Vaccines is directly oral, is also through extracting, being processed into oral or ejection preparation use by the proteantigen of expression of plants.

This technical scheme has following beneficial effect:

1, the present invention is more safer than traditional immunization routes, does not need the equipment of syringe needle and syringe and so on.Syringe needle and syringe not only spend fund, and dangerous.If used not through syringe needle and the syringe of sterilization, propagate blood-borne disease likely via them.Other vaccines are cultivated at mass cell or in reproductive process, be easy to the pollution that pathogenic microorganism particularly Mycoplasma occurs, and Transgenic Plant Vaccines are not existed this problem, because plant virus not infection animal.

2, the present invention is more more convenient than traditional immunization routes, directly oral.Traditional immunization routes has to pass through non-oral routes inoculation and body could be induced to produce cyclicity IgG antibody, is difficult to produce secretory IgA local antibody, therefore can not stop that toxin enters body from mucosal absorption, and oral immunity can produces secretory IgA.

3, the present invention not only provides vaccine, and provides nutrition.Utilize the oral vaccine that transgenic plant is produced, provide not only antigen protein, and provide abundant nutrient substance, contribute to after animal edible improving immunity of organisms.

4, the present invention is more effective than traditional immunization routes.Can be subject to the protection of cell wall during the sour environment of plant vaccine antigen by gastric, directly arrive intestinal mucosal inductive site, stimulating mucosal and systemic immune response, traditional parenteral vaccine almost can not produce special mucosal immunity.Between pathogen and host, its effectiveness directly can be improved greatly with antigen induced immunoreation in interactional initial position.

5, the present invention is easier to store and distribution than traditional vaccine, does not need cold preservation and low-temperature transport.Not only save refrigerating equipment, reduce cost, and easy to use, can long term administration at any time, administration process is very convenient, avoids loaded down with trivial details immune programme for children.And compared with traditional vaccine, administration range is wider, both can be used for people and domestic animal, and also can be used for wild a vegetarian animal.

6, production cost of the present invention is cheaper, does not need complicated armarium, easy large-scale production.After vaccine antigen gene proceeds to edible plant, the place that only need be suitable for, water, fertilizer and pesticide just can large-scale production on a small quantity, do not need to resemble traditional mode of production vaccine and carry out strict separation and purification.Can obtain large quantities of plant vaccine by simple asexual propagation, save many instrument and equipments, production cost significantly reduces, for the developing country of the shortage of capital, and especially individual good news.

7, the present invention is compared with other vaccines, has complete eukaryotic expression system, makes expression product have good immunogenicity and biological activity.Transgenic plant can carry out post translational processing accurately to albumen mass-energy and modify, and make three-D space structure more be tending towards naturalness, the antigen of expression has similar immunogenicity and biological activity to infection animal antigen.

8, the present invention constructs welch's fusiform bacillus alpha toxin gene plant expression vector pA, construct LTB (LT-B) and welch's fusiform bacillus alpha toxin fusion gene plant expression vector pLA simultaneously, agrobacterium tumefaciens LBA 4404 is successfully proceeded to by freeze-thaw method, obtained by agrobacterium-mediated transformation and transform Herba Medicaginis, through Basta herbicide screening, obtain Herbicid resistant Herba Medicaginis to identify through PCR detection, PCR primer order-checking, SDS-PAGE and Western blot, prove that transgenic alfalfa have expressed genes of interest albumen.

9, the transgenic alfalfa of antigen expressed albumen is carried out asexual propagation, obtain clone three generations altogether, identify through 0.02%Basta herbicide screening, PCR and Western blot, prove that recombinant antigen 100% can clone stably express in offspring and heredity at transgenic alfalfa.

10, with pA and pLA transgenic alfalfa oral immunity origin animal rabbit and mice, result shows that two kinds of transgenic alfalfa expression systems all can induce origin animal to produce serum antibody IgG, irritates nucous membrane system secretion IgA antibody, and has immunogenicity; Alpha toxin with mucosal adjuvant LT-B has better immunogenicity; The alpha toxin antigen protein safety non-toxic that transgenic alfalfa is expressed; body can be stimulated to produce immanoprotection action; resist the strong virus attack of pathogenic A type clostridium perfringens alpha toxin; induction body produce IgG serum antibody can effectively in and A type bacillus perfringens secrete natural toxin, for the research and development of transgene plant oral vaccine provide material base.

Accompanying drawing illustrates:

Accompanying drawing 1 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgG level in immune rabbit serum.

Accompanying drawing 2 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgG levels in immune serum.

Accompanying drawing 3 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgA levels in immune rabbit feces.

Accompanying drawing 4 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgA levels in immune mouse feces.

Accompanying drawing 5 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgA levels in the outer reproductive tract flushing liquor of immune rabbit.

Accompanying drawing 6 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgA levels in the outer reproductive tract flushing liquor of immune mouse.

Accompanying drawing 7 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgA levels in immune rabbit nasal wash.

Accompanying drawing 8 is specificity anti-A type welch's fusiform bacillus alpha toxin protein IgA levels in immune mouse nasal wash.

Accompanying drawing 9 is specificity anti-A type welch's fusiform bacillus alpha toxin protein sIgA levels in immune mouse AK-Rinse.

Accompanying drawing 10 is specificity anti-A type welch's fusiform bacillus alpha toxin protein sIgA levels in immune mouse intestinal mucus.

Detailed description of the invention:

Embodiment 1:

The manufacture method of anti-clostridieum welchii disease Transgenic Plant Vaccines, the method comprises:

(1) with containing the plasmid of coding clostridieum welchii alpha toxin or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain welch's fusiform bacillus alpha toxin gene, the welch's fusiform bacillus alpha toxin gene of acquisition and Ti-plasmids are carried out enzyme action respectively, reclaim genes of interest and plant expression vector fragment, connect and transformation of E. coli, enzyme action is carried out to restructuring Ti, PCR and order-checking qualification, use recombinant plasmid transformed agrobacterium tumefaciens, screening, PCR qualification obtains positive restructuring agrobacterium tumefaciens pA: be preserved in China typical culture collection center on June 2nd, 2010, preserving number is: CCTCC NO:M 2010130.

(2) plant seed is through vernalization, sprouting, montage, obtain explant, explant is infected respectively with the agrobacterium tumefaciens pA of restructuring in step (1), by screening, subculture, take root, transplant, obtain transformed plant, Screening and Identification transgenic positive plant, extracts transgenic plant genome or albumen carries out Southern blot, Northern blot or Western blot identifies, proves that clostridieum welchii toxin is expressed in plant.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, the clostridieum welchii alpha toxin described in step (1) also can adopt clostridieum welchii alpha toxin and/or clostridieum welchii β toxin and/or clostridieum welchii ε toxin and/or clostridieum welchii ι toxin; Described Ti-plasmids also can adopt Ri plasmid, and the agrobacterium tumefaciens simultaneously adopts Agrobacterium rhizogenes.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, plant described in step (2) comprises herbage or vegetable or crops or fruit or model plant, described herbage comprises Herba Medicaginis, described vegetable comprises Rhizoma Solani tuber osi, described crops comprise Semen Maydis, described fruit comprises Fructus Lycopersici esculenti, and described model plant comprises Nicotiana tabacum L..

Utilize the anti-clostridieum welchii disease Transgenic Plant Vaccines that above-mentioned method makes, described anti-clostridieum welchii disease Transgenic Plant Vaccines is directly oral, is also through extracting, being processed into oral or ejection preparation use by the proteantigen of expression of plants.

Embodiment 2:

A manufacture method for anti-clostridieum welchii disease Transgenic Plant Vaccines, the method comprises:

(1) with containing the plasmid of mucosal adjuvant LTB (LT-B) or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain mucosal adjuvant LT-B gene, with containing the plasmid of coding clostridieum welchii alpha toxin or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain welch's fusiform bacillus alpha toxin gene, described mucosal adjuvant LT-B gene and described welch's fusiform bacillus alpha toxin gene are merged and obtains fusion gene, acquisition fusion gene and Ti-plasmids are carried out enzyme action respectively, reclaim genes of interest and plant expression vector fragment, connect and transformation of E. coli, enzyme action is carried out to restructuring Ti-plasmids, PCR and order-checking qualification, use recombinant plasmid transformed agrobacterium tumefaciens, screening, PCR qualification obtains positive restructuring agrobacterium tumefaciens pLA: be preserved in China typical culture collection center on June 2nd, 2010, preserving number is: CCTCC NO:M 2010131.

(2) plant seed is through vernalization, sprouting, montage, obtain explant, explant is infected respectively with the agrobacterium tumefaciens pLA of restructuring in step (1), by screening, subculture, take root, transplant, obtain transformed plant, Screening and Identification transgenic positive plant, extracts transgenic plant genome or albumen carries out Southern blot, Northern blot or Western blot identifies, proves that clostridieum welchii toxin is expressed in plant.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, the LTB (LT-B) described in step (1) also can adopt b subunit of cholera toxin (CT-B).

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, the clostridieum welchii alpha toxin described in step (1) also can adopt clostridieum welchii alpha toxin and/or clostridieum welchii β toxin and/or clostridieum welchii ε toxin and/or clostridieum welchii ι toxin; Described Ti-plasmids also can adopt Ri plasmid, and the agrobacterium tumefaciens simultaneously adopts Agrobacterium rhizogenes.

The manufacture method of described anti-clostridieum welchii disease Transgenic Plant Vaccines, plant described in step (2) comprises herbage or vegetable or crops or fruit or model plant, described herbage comprises Herba Medicaginis, described vegetable comprises Rhizoma Solani tuber osi, described crops comprise Semen Maydis, described fruit comprises Fructus Lycopersici esculenti, and described model plant comprises Nicotiana tabacum L..

The anti-clostridieum welchii disease Transgenic Plant Vaccines utilizing said method to make, described anti-clostridieum welchii disease Transgenic Plant Vaccines is directly oral, is also through extracting, being processed into oral or ejection preparation use by the proteantigen of expression of plants.

Embodiment 3:

1 test material

1.1 plasmids and strain

Plasmid pET-30b-a contains welch's fusiform bacillus alpha toxin gene sequence, is built by this laboratory; Plasmid pLT-b contains LTB (LT-B) gene order, is built by this laboratory teacher Ge Junwei; Expression of plants type carrier p33EG contains E12 promoter sequence, the Ω sequence strengthening translation, the Bar gene of anti-PPT (Phosphinothricin) herbicide, prokaryotic expression Npt II (Km resistance) gene and agrobacterium tumefaciens LBA 4404 and is awarded by Northeast Agricultural University's Life Science College Zhu Yan penetrating judgment and be so kind as to give; A type clostridieum welchii (C57-1) is purchased from China Veterinery Drug Inspection Office.

1.2 antibody preparation

Anti-clostridieum welchii alpha toxin rabbit anteserum, mice serum and anti-LTB (LT-B) mice serum are prepared by this laboratory; The goat anti-rabbit igg of goat-anti rabbit IgA, HRP labelling of the sheep anti-mouse igg of HRP labelling, sheep anti mouse IgA, HRP labelling of HRP labelling is all purchased from sigma company.

1.3 enzyme preparation

The restricted enzyme such as rTaq archaeal dna polymerase, Ex Taq archaeal dna polymerase, T4DNA ligase, BmaHI, SacI, XbaI is purchased from the precious biological engineering company limited in Dalian.

1.4 biochemical reagents and test kit

DNA MarkerDL2000, DNA MarkerDL15000, pre-dyed Protein Marker (118 ~ 19kDa), Protein Marker (116.0 ~ 14.0kDa) etc. are purchased from the precious biological engineering company limited in Dalian; Mini-scale plasmid DNA extraction kit, in a small amount glue reclaim test kit purchased from Shanghai Hua Shun biological engineering company limited; The biochemical reagents available from Sigma such as CTAB, 6-Benzylaminopurine (6-BA), IBA, Basta, Glufosinate-ammonium; Kanamycin (Km), streptomycin (Sm), rifampicin (Rif) etc. are purchased from Beijing Suo Laibao biotech firm.

1.5 experimental animals and plant

BALB/c mouse in 6 ~ 8 week age (16 ± 2g) is cleaning grade, purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture; About 45 days non-immunizing rabbits (1 ± 0.2kg), purchased from Second Affiliated Hospital of Harbin Medical Univ..

Zhaodong alfalfa seed is awarded by Northeast Agricultural University's Zhu Yan penetrating judgment and is so kind as to give.

1.6 primer synthesis

With plasmid pET-30b-a (containing coding welch's fusiform bacillus alpha toxin gene) for template, according to the preferences that plant expression vector restriction enzyme site and vegetable codon use, application oligo6.0 software design pair of primers P1 and P2 is as shown in table 1-1:

Table 1-1 primer sequence

Underscore part is the restriction enzyme site of design: P1, P3 introduce BamHI, P2 introducing SacI, P4, P5 introduces XbaI;

Blocked portion is: P1, P3 introduce Kozak sequence, and P2 introduces SEKDEL sequence, and P4 introduces Linker sequence.

With plasmid pLT-b (LTB) for template, according to plant expression vector, the preferences connecting alpha toxin gene restriction enzyme site and vegetable codon use, Design Internet Applications pair of primers P3 and P4; With plasmid pET-30b-a for template, according to plant expression vector and connection LT-B toxin gene restriction enzyme site, Design Internet Applications forward primer P5; With alpha toxin portion gene for template, Design Internet Applications a pair is for detecting primer P6 and P7 of this gene expression.

2 test methods

The structure of 2.1 welch's fusiform bacillus alpha toxin gene plant expression vectors

2.1.1PCR amplification alpha toxin gene

(1) extraction of pET-30b-a plasmid

Adopt LB plate streak activation-70 DEG C of strain BL21 (DE3) pET-30b-a preserved, picking list bacterium colony, be inoculated in 5mL containing in the LB culture medium of 50mg/LKm, 220r/min 37 DEG C of overnight incubation.Utilize plasmid DNA Mini Kit to extract plasmid pET-30b-a ,-20 DEG C save backup.

(2) acquisition of alpha toxin gene

With pET-30b-a plasmid for masterplate, P1 and P2 is primer, pcr amplification alpha toxin genes of interest

Reaction cumulative volume 50.0 μ L

PET-30b-a template 1 μ L

EX Taq archaeal dna polymerase 0.5 μ L

10×EX PCR Buffer 5μL

Primer P1 (25pmol/ μ L) 1 μ L

Primer P2 (25pmol/ μ L) 1 μ L

dNTP 4μL

Deionized water 37.5 μ L

Reaction condition is: enter PCR circulation after 95 DEG C of denaturation 5min, 94 DEG C of degeneration 1min, 53.5 DEG C of annealing 1min, and 72 DEG C extend 1min, 30 circulations, and last 72 DEG C extend 10min.0.8% agarose gel electrophoresis, Ultraviolet Detector observes pcr amplification result: the alpha toxin genes of interest fragment obtaining about 930bp.

2.1.2 the connection of alpha toxin gene and plant expression vector

(1) double digestion of plant expression vector and genes of interest

Plant expression vector is Ti-plasmids, adopts LB plate streak activation-70 DEG C of strain p33EG/JM109 preserved, picking list bacterium colony, is inoculated in 5mL containing in the LB culture medium of 50mg/L Km, 220r/min37 DEG C of overnight incubation.Plasmid DNA Mini Kit is utilized to extract p33EG plasmid.Get the PCR primer of 25 μ L alpha toxin genes of interest, add the centrifugal 10min of 50 μ L dehydrated alcohol precipitation 30min, 12000r/min, abandon supernatant, add 70% ethanol 200 μ L and wash precipitation 2 times, after drying to be precipitated, get p33EG plasmid 7 μ L simultaneously and carry out double digestion by following system.

PCR primer precipitation p33EG plasmid 7 μ L

BamH I 1μL BamH I 1μL

Sac I 1μL Sac I 1μL

10×KBuffer 1μL 10×K Buffer 1μL

ddH ₂O 17μL ddH ₂O 10μL

BamH I/SacI double digestion is totally 20 μ L, 37 DEG C of water-bath 4h, PCR and p33EG plasmid double digestion product mixes with 10 × Loading Buffer3 μ L respectively, gets 2 μ L samples and carries out 1% agarose gel electrophoresis, observed result: p33EG obtains about 12500bp and 1800bp genetic fragment.

(2) glue reclaims and is connected

After electrophoresis terminates, cut the gel containing object fragment respectively, carry out recovery with miniprep dna glue recovery test kit and purify, reclaim product-20 DEG C and save backup.The alpha toxin gene PCR digestion products reclaiming test kit recovery through glue is connected with plant expression vector p33EG plasmid enzyme restriction large fragment, and coupled reaction system is as follows:

Total system 20 μ L

P33EG plasmid enzyme restriction large fragment 2 μ L

Alpha toxin gene PCR digestion products 6 μ L

T4 DNALigase 1μL

10×Ligase Buffer 2μL

ddH ₂O 9μL

Gently after mixing, be placed on and connect in instrument, 16 DEG C of water-baths connect spends the night, and connects product and is used for transformation of E. coli JM109 competent cell.Bacterium colony in the some LB transformation plate of random picking, be inoculated in 5mL containing in the LB culture medium of 50mg/LKm, 220r/min 37 DEG C of overnight incubation, extract plasmid for subsequent use.

(3) selection systems of recombinant plant expression vector

Carry out the single, double enzyme action qualification of BamHI and SacI and PCR and order-checking to recombiant plasmid pa to identify.

1. single endonuclease digestion qualification

BamHI single endonuclease digestion system SacI single endonuclease digestion system

Pa plasmid 2 μ L pa plasmid 2 μ L

BamHI 1μL SacI 1μL

10×K Buffer 1μL 10×L Buffer 1μL

ddH ₂O 6μL ddH ₂O 6μL

Single endonuclease digestion is totally 10 μ L, 37 DEG C of water-bath 2h, 0.8% agarose gel electrophoresis, observed result: obtain about 13430bp object fragment.

2. double digestion qualification

Pa plasmid 4 μ L

BamHI 1μL

SacI 1μL

10×K Buffer 1μL

ddH ₂O 13μL

Double digestion is totally 20 μ L, 37 DEG C of water-bath 4h, and 0.8% agarose gel electrophoresis observes enzyme action result: obtain about 12500bp and 930bp genes of interest fragment.

3. PCR qualification (total system 25 μ L)

Pa template 0.5 μ L

RTaq archaeal dna polymerase 0.5 μ L

10×PCR Buffer 2.5μL

Primer P1 (25pmol/ μ L) 0.5 μ L

Primer P2 (25pmol/ μ L) 0.5 μ L

dNTP 2.0μL

Deionized water 18.5 μ L

Reaction condition is: 95 DEG C of denaturation 5min, 94 DEG C of degeneration 1min, 53.5 DEG C of annealing 1min, and 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min, 0.8% agarose gel electrophoresis, observe PCR result: obtain about 930bp genes of interest fragment.

4. to check order qualification

Enzyme action qualification and PCR qualification are positive bacterial strain check order, sequencing result is completely the same with expection.

2.1.3LT-B with the connection of alpha toxin fusion gene and plant expression vector

(1) acquisition of genes of interest

With pLT-b plasmid for masterplate, P3 and P4 is primer, pcr amplification LT-B genes of interest:

Reaction cumulative volume 50.0 μ L

PLT-b template 1 μ L

EX Taq archaeal dna polymerase 0.5 μ L

10×EX PCR Buffer 5μL

Primer P3 (25pmol/ μ L) 1 μ L

Primer P4 (25pmol/ μ L) 1 μ L

dNTP 4μL

Deionized water 37.5 μ L

Reaction condition is: 95 DEG C of denaturation 5min, 94 DEG C of degeneration 30s, 47.5 DEG C of annealing 30s, and 72 DEG C extend 30s, 30 circulations, and 72 DEG C extend 10min.1.5% agarose gel electrophoresis, observed result: the LT-B genes of interest fragment obtaining about 340bp.

With pET-30b-a plasmid for masterplate, P5 and P2 is both sides primer, pcr amplification alpha toxin genes of interest:

Reaction cumulative volume 50.0 μ L

PET-30b-a template 1 μ L

EX Taq archaeal dna polymerase 0.5 μ L

10×EX PCR Buffer 5μL

Primer P5 (25pmol/ μ L) 1 μ L

Primer P2 (25pmol/ μ L) 1 μ L

dNTP 4μL

Deionized water 37.5 μ L

Reaction condition is: 95 DEG C of denaturation 5min, 94 DEG C of degeneration 1min, 53.5 DEG C of annealing 1min, and 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min.0.8% agarose gel electrophoresis, observed result: the alpha toxin genes of interest fragment obtaining about 930bp.

(2) genes of interest and plant expression vector enzyme action be connected

Get the PCR primer mixing of 7 μ LT-B and alpha toxin gene respectively, add the centrifugal 10min of 30 μ L dehydrated alcohol precipitation 30min, 12000r/min, abandon supernatant, add 70% ethanol 100 μ L and wash precipitation 2 times, after drying to be precipitated, carry out BamH I, Xba I and Sac I tri-enzyme action by following system.

Genes of interest PCR primer precipitates

BamHI 1μL

XbaI 1μL

SacI 1μL

10×K Buffer 1μL

ddH ₂O 16μL

Three enzyme action are totally 20 μ L, 37 DEG C of water-bath 4h, PCR digestion products all carries out 1% agarose gel electrophoresis, after electrophoresis terminates, cut the gel containing object fragment respectively, carry out recovery with miniprep dna glue recovery test kit to purify, the LT-B reclaiming test kit recovery through glue is connected with plant expression vector p33EG plasmid double digestion large fragment with alpha toxin gene PCR three digestion products, and 20 μ L coupled reaction systems are as follows:

P33EG plasmid double digestion large fragment 2 μ L

LT-B gene PCR three digestion products 3 μ L

Alpha toxin gene PCR three digestion products 3 μ L

T4 DNA Ligase 1μL

10×Ligase Buffer 2μL

ddH ₂O 9μL

Gently after mixing, be placed on and connect in instrument, 16 DEG C of water-baths connect spends the night, and connects product and is used for transformation of E. coli JM109 competent cell.Bacterium colony in the some LB transformation plate of random picking, is inoculated in 5mL containing in the LB culture medium of 50mg/LKm, 220r/min 37 DEG C of overnight incubation, utilizes test kit to extract plasmid.

(3) qualification of recombinant plant expression vector

Carry out the single, double enzyme action qualification of BamHI and SacI to recombiant plasmid pla, PCR identifies total system (50 μ L):

Pla template 1 μ L

RTaq archaeal dna polymerase 0.5 μ L

10×PCR Buffer 5μL

Primer P3 (25pmol/ μ L) 1 μ L

Primer P2 (25pmol/ μ L) 1 μ L

dNTP 4μL

ddH ₂O 37.5μL

Reaction condition is: 95 DEG C of denaturation 5min, 94 DEG C of degeneration 1min, 50.5 DEG C of annealing 1min, and 72 DEG C extend 1min, 30 circulations, 72 DEG C of extension 10min.1% agarose gel electrophoresis observed result: pla, through BamHI and SacI single endonuclease digestion, obtains about 13770bp object fragment; Pla, through BamHI and SacII double digestion, obtains about 12500bp and 1270bp genetic fragment; Pcr amplification obtains about 1270bp object fragment.

Order-checking qualification: enzyme action qualification and PCR qualification are positive bacterial strain order-checking, and result is consistent with expection.

The qualification of 2.2 alpha toxin gene transformation Herba Medicaginiss and expressing protein thereof

2.2.1 genes of interest freeze-thaw method transformation Agrobacterium

(1) Agrobacterium competent cell is prepared

The Agrobacterium LBA4404 bacterium lyolysis that-70 DEG C are preserved frozen, line connects bacterium in YEB solid medium; The single colony inoculation of picking Agrobacterium contains in the YEB fluid medium of 50mg/LSm, 50mg/L Rif in 5mL, 28 DEG C, 200r/min shaken cultivation spends the night; Above-mentioned bacterium liquid is inoculated in 50mL in 1: 10 ratio and contains continuation cultivation in corresponding antibiotic YEB fluid medium, until OD ₆₀₀be about 0.5; 4 DEG C, the centrifugal 5min of 5000r/min, abandon supernatant; Proceed to sterile centrifugation tube, use 20mmol/L CaCl ₂the resuspended thalline of 100 μ L, ice bath 30min; 4 DEG C, the centrifugal 5min of 5000r/min, abandon supernatant; With the CaCl that 1mL is cold ₂(20mmol/L) suspend, be distributed into 100 μ L/ and manage, add 40% glycerol of 100 μ L pre-coolings, to-70 DEG C of preservations after liquid nitrogen flash freezer.

(2) freeze-thaw method transformation Agrobacterium

Get genes of interest recombiant plasmid pa and pla that 1 μ L purifies, add respectively in 100 μ L LBA4404 Agrobacterium competent cells and mix; Ice bath 5min, liquid nitrogen freezing 8min, rapidly to 37 DEG C temperature bath 8min; Add YEB fluid medium 600 μ L, 28 DEG C, 200r/min expresses 4 ~ 5h in advance; The centrifugal 5min of 3000r/min, removes most of supernatant, is gone to by residue about 50 μ L bacterium liquid pipettors on the YEB solid medium containing 100mg/LKm, 50mg/LSm, 50mg/LRif and smoothens; 28 DEG C, 200r/min, cultivate 48 ~ 96h.

(3) PCR screening and identification recombinational agrobacterium

With single bacterium colony that freeze-thaw method transformation plate grows for template, pcr amplification is carried out with genes of interest Auele Specific Primer, recombiant plasmid pla, with 2.1, by the Agrobacterium called after pA of the recombinant plasmid transformed positive, is transformed positive Agrobacterium called after pLA by PCR reaction system and program.

2.2.2 genes of interest Agrobacterium-mediated transformation Herba Medicaginis

(1) cultivation of explant

The sterilization of seed and vernalization: selected alfalfa seed is fully washed, 70% alcohol disinfecting 1min, 0.1% mercuric chloride surface sterilization 15min, two tweens are added after outwelling waste liquid, on shaking table, 105r/min shakes 15min, with aseptic water washing 4 ~ 5 times, does not finally have seed with water logging, more than 48h is preserved in 4 DEG C of refrigerators, take out, be inoculated on seed germination medium, condition of culture is 26 DEG C, 1500lx, 16h/d illumination.Then be the shearing of cotyledonary node: 6 ~ 7d after inoculation, treat that cotyledon launches completely, under cotyledonary node, 1mm place cuts, and tear two panels cotyledon and be inoculated on adventitious bud induction culture base, preculture 3 ~ 4d, can infect when material cut place just expands.

(2) the infecting of Agrobacterium

Be inoculated in by pA and pLA recombinational agrobacterium on the YEB solid medium of additional 100mg/LKm, 50mg/L Sm, 50mg/LRif, 26 DEG C of light culture are to growing single bacterium colony.With connecing collarium picking list bacterium colony, be seeded in 5mL containing in corresponding antibiotic YEB fluid medium, 200r/min 28 DEG C of overnight incubation, treat that bacterium liquid grows to OD ₆₀₀when being 0.4 ~ 0.6, the ratio in 1: 10 is inoculated in shaken cultivation in fresh above-mentioned culture medium, carries out re-activation.Work as OD ₆₀₀when being 0.5 ~ 0.6, draw bacterium liquid in sterile centrifugation tube, the centrifugal 10min of 2000r/min, removes supernatant, resuspended, for subsequent use with isopyknic YEB fluid medium.The explant getting preculture 3 ~ 4d, in aseptic triangular flask, adds bacterium liquid resuspended in right amount, 150r/min, 28 DEG C, infects 15min.The explant infected is seeded in the bud inducement culture medium of additional 100mg/L Awei acid, at 26 DEG C of dark place Dual culture 3 ~ 4d, is as the criterion with naked eyes visible colonies.

(3) cultivation of degerming and seedling

Learnt from else's experience the explant of Dual culture in aseptic triangular flask, by the liquid wash 3 ~ 4 times of additional 100mg/L amoxicillin sodium and clavulanate potassium bacteria remover, then transferred in the degerming screening culture medium of additional 0.5mg/LGlufosinate-ammonium.Condition of culture is 26 DEG C, 1500lx, 16h/d illumination.Screening and culturing is after 2 weeks, and the transformant of explant will differentiate resistant buds, is proceeded in corresponding Elongation of adventitious bud culture medium and carries out successive transfer culture.By the seedling through successive transfer culture, implant in root media and cultivate, until take root.By take root, the aseptic seedling that grows to bottleneck, be transferred to the peat composed of rotten mosses: soil=1: cultivate in the Seedling alms bowl of 3, grow to and be to a certain degree transplanted in flowerpot afterwards, continue to cultivate in greenhouse, finally obtain 238 strains and transform alfalfa plants, Herba Medicaginis 131 strain of its transfer pA, turns Herba Medicaginis 107 strain of pLA.

2.2.3 herbicide basta screening transforms Herba Medicaginis

(1) the screening determination of Basta concentration

18% commercial Basta herbicide adds deionized water and is mixed with gradient concentration: 0.07%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%.With non-transgenic alfalfa plants for contrast, blade will be smeared respectively the Basta of variable concentrations, observed result after 2 days: serious phytotoxicity reaction all appears in the blade smearing 0.07%, 0.05% and 0.04%, and excessive concentration is described, is unsuitable for screening transgenic Herba Medicaginis; The blade smearing 0.01% does not react, and illustrates that concentration is too low, is also unsuitable for screening transgenic Herba Medicaginis; Smear 0.03% and 0.02% blade phytotoxicity reaction moderate, tentatively determine that the screening concentration of herbicide basta is 0.03%.

(2) Basta screening transforms Herba Medicaginis

18% commercial Basta herbicide formulation is become the concentration of 0.02%, utilizes aerosol apparatus even application to transform Herba Medicaginis, simultaneously with non-transgenic alfalfa for contrast, the Herba Medicaginis survived is considered as transgenic alfalfa.0.03% commercial Basta herbicide tentatively sprays 38 strains and transforms Herba Medicaginiss, simultaneously with non-transgenic alfalfa for contrast.Spray latter 7 days, non-transgenic alfalfa is withered jaundice rapidly, and stem and the leaf of most conversion Herba Medicaginis also all occur serious phytotoxicity reaction, only have minority Herba Medicaginis still to keep green.Spray latter 15 days, the stem and the leaf that transform Herba Medicaginis are almost all dried-up, and green Herba Medicaginis also withers because of the further effect of poisoning, and the screening concentration of visible 0.03% is still too high, determine that remaining 200 strains of screening concentration spraying of employing 0.02% transform Herba Medicaginis.0.02% commercial Basta herbicide sprays 200 strains and transforms Herba Medicaginiss, simultaneously with non-transgenic alfalfa for contrast.Spray latter 15 days, non-transgenic alfalfa withered death gradually, what transform that the stem of Herba Medicaginis and leaf also have in various degree is withered, but the overwhelming majority transforms Herba Medicaginis can sprout tender shoots very soon, grows up to new plant further; Only have minority part equally whole withered with non-transgenic alfalfa, last dead because of butt rot.The Herba Medicaginis survived is considered as antiweed and transforms Herba Medicaginis, obtain 201 strain transgenic alfalfas altogether, 116 strains of its transfer pA, turn 85 strains of pLA.

2.2.4PCR screening and identification transgenic alfalfa

(1) CTAB method extracts sativa genomic dna

Get 0.05g fresh alfalfa blade in 1.5mL centrifuge tube, liquid nitrogen grinding.Add 2 × CTAB900mL of preheating 65 DEG C, 65 DEG C of water-bath 20min, shake up once every 2min, take out and are cooled to room temperature.Add chloroform: isoamyl alcohol (24: 1) 500mL mixing shakes up, and 4 DEG C, 7500r/min, centrifugal 10min, come again.Get supernatant, add 1/10 volume 3mol/LNaAC and isopyknic isopropyl alcohol, jog occurs to flocculent deposit.Centrifugal, abandon supernatant, with 70% ethanol purge 2 times, drying at room temperature 1h, precipitation is dissolved in 50 μ L sterile deionized water.

(2) PCR screening and order-checking qualification transgenic alfalfa

With transgenic alfalfa genomic DNA for template, carry out pcr amplification with alpha toxin gene versatility primer:

Cumulative volume 25.0 μ L

Transgenic alfalfa genomic DNA 0.5 μ L

RTaq archaeal dna polymerase 0.5 μ L

10×PCR Buffer 2.5μL

Primer P6 (25pmol/ μ L) 0.5 μ L

Primer P7 (25pmol/ μ L) 0.5 μ L

dNTP 2.0μL

MgCl ₂(25mmol/L) 2.5μL

ddH ₂O 16.0μL

Randomly draw pA and pLA and transform each 30 strains of Herba Medicaginis, with its genome for template carries out PCR screening and identification, reaction condition is: 95 DEG C of denaturation 5min, 94 DEG C of degeneration 1min, 52.8 DEG C of annealing 1min, and 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min.0.8% agarose gel electrophoresis observes PCR result: obtain 51 strain alpha toxin positive transgenic Herba Medicaginiss altogether, 24 strains of its transfer pA positive, turn 27 strains of the pLA positive.With 27 strain Herba Medicaginiss for template, carry out the detection of LT-B genes of interest, result obtains the positive Herba Medicaginis of 21 strain PCR.The PCR primer of positive plant checked order, sequencing result is consistent with expection.

2.2.5 the qualification of Herba Medicaginis express alpha toxin protein

(1) SDS-PAGE of Herba Medicaginis expressing protein analyzes

Induction expression of recombinant e. coli alpha toxin albumen: picking single restructuring bacterium colony BL21 (DE3) pET-30b-a, be inoculated in the LB culture medium containing 50mg/LKm, 37 DEG C of shaken cultivation are spent the night, next day, and 1% inoculation is containing in the LB culture medium of 50mg/LKm.37 DEG C are cultured to OD ₆₀₀when being about 0.5, add IPTG to final concentration 0.4mmol/L, 37 DEG C of inducing culture 2h.The centrifugal 5min of all samples 8000r/min, 100 μ L PBS are resuspended, add 2 × SDS sample-loading buffer (containing DTT) 100 μ L, fully mix, boil 10min, carry out SDS-PAGE electrophoresis.

Extract Herba Medicaginis expressing protein: get and contrast non-transgenic alfalfa and PCR and be accredited as the positive each 0.5g of transgenic alfalfa blade and put in Potter-Elvehjem Tissue Grinders, non-transgenic alfalfa albumen compares, add 1mL vegetable protein extracting solution, ice bath, rapid grinding, be collected in 1.5mL centrifuge tube, 4 DEG C, 12000r/min, centrifugal 20min.Get 50 μ L supernatants, add 2 × SDS sample-loading buffer (containing DTT) 50 μ L, fully mix, boil 10min, protein standard marker is reference, carries out SDS-PAGE electrophoresis.

10 μ L samples add in sample well, with protein standard marker as a reference, carry out electrophoresis, and initial voltage is 90V, to be instructed dose when entering separation gel, and voltage is increased to 120V, terminates to electrophoresis; Take out gel, after coomassie brilliant blue staining, decolouring, observe destination protein expression effect: recombinant bacterium BL21 (DE3) pET-30b-a induces bacterium liquid and pA transgenic alfalfa albumen all to have the alpha toxin albumen of about 35kD to swim through IPTG and is with, and non-transgenic alfalfa albumen obtains without, preliminary proof destination protein and expresses.

(2) the Western blot of Herba Medicaginis expressing protein identifies

The alpha toxin albumen of expression of recombinant e. coli and alfalfa are after SDS-PAGE analyzes, further with the expression of Western blot method qualification destination protein, primary antibodie is the rabbit anti-clostridieum welchii alpha toxin serum of 1: 100 dilution, two to resist be the goat anti-rabbit igg of HRP labelling of 1: 3000 dilution, observe colour developing result: all occur obvious color reaction in the desired location of expressing protein, illustrate utilize the albumen of Expressed in Transgenic Plant also can identify by welch's fusiform bacillus alpha toxin antibody in rabbit anteserum, show that recombinant alpha toxin protein obtains effective expression in Herba Medicaginis, and the albumen of expressing equally with prokaryotic expression protein has antigenic specificity.

The research of 2.3 alpha toxin transgenic alfalfa hereditary stabilityes

2.3.1 the clone of transgenic alfalfa

Choose PCR qualification, order-checking qualification and Western blot qualification and be positive each 1 strain of pA and pLA transgenic alfalfa, select each 3 healthy and strong of branch and cut with shears, be inserted in liquid root media and carry out root culture.Cross about 20 days roots to generate, implant in Seedling alms bowl and continue cultivation, plant in flowerpot after a period of time when root is strong, thus obtain a transgenic alfalfa clone generation, same acquisition clones secondary and clone three generations.

2.3.2 the screening and identification of transgenic alfalfa clone offspring

After transgenic alfalfa clone offspring grows to certain altitude, carry out 0.02%Basta screening, PCR qualification, 100% is positive as a result.By being accredited as, each 5 μ L of positive pA and pLA transgenic alfalfa generation protein sample mix loadings, secondary and three generations's sample mixes loading equally further, with LT-B and α of 1: 100 dilution-toxin mixing mice hyper-immune serum for primary antibodie, the sheep anti-mouse igg of 1: 3000 dilution HRP labelling is two to resist, and carries out Western blot qualification.There is the immune response belt of two expection sizes in result: the alpha toxin protein band of about 35kD all appears in pA positive Herba Medicaginis clone offspring, all there is the LTB-alpha toxin fusion rotein band of about 45kD in pLA positive Herba Medicaginis clone offspring, and non-transgenic alfalfa albumen reacts without this, prove that the transgenic alfalfa clone offspring obtained by asexual propagation has stable inherited character.

The mensuration of 2.4 oral administration of alpha toxin transgene Herba Medicaginis amynologic index

2.4.1 alpha toxin transgenic alfalfa immunity test animal

(1) the oral immunity preparation of Herba Medicaginis

The preparation of Herba Medicaginis juice: the fresh blade gathering pA and pLA transgenic alfalfa and non-transgenic alfalfa, precise, adds PBS solution to 0.4g/mL respectively, tissue mashing machine is stirred to scattered paste shape, eight layers of filtered through gauze, now by existing system during each immunity, filtered juice 4 DEG C is for subsequent use.

The preparation of Herba Medicaginis rabbit grain: the fresh blade and the twig that gather pA and pLA transgenic alfalfa and non-transgenic alfalfa, shady and cool place natural air drying, Chinese medicine grinder is crushed to Powdered, correct amount, ratio in 1: 10 is added in common rabbit grain respectively, for subsequent use after dry.

(2) test mice grouping and immunity thereof

Test Mus is divided into 3 groups, often organizes 24 (male and female half and half).Test and gavage the Herba Medicaginis juice that Herba Medicaginis juice 200 μ L (0.08g/ day fresh leaf) turns pA I group every mice day; Test the Herba Medicaginis juice that II group of oral Isodose turns pLA; The not genetically modified Herba Medicaginis juice of the test oral Isodose of III group, each continuous immunity 7d, every day 1 time, altogether immunity five times, 1 week, first three secondary immunization time interval, 3 weeks, rear twice immunization time interval.

(3) rabbit grouping and immunity thereof is tested

Test rabbit is divided into 3 groups, often organizes 6 (being doe).Test I group of every rabbit 100g (200g/ day fresh leaf) that feeds and turn the Herba Medicaginis rabbit grain of pA, every day 1 time; Test the Herba Medicaginis rabbit grain that II group of Isodose of feeding turns pLA; Test III group is fed Isodose not genetically modified Herba Medicaginis rabbit grain, each continuous immunity 7d, altogether immunity three times, 1 week, each immunization time interval; After again immunity twice, by feeding, Herba Medicaginis rabbit grain changes oral Herba Medicaginis juice into, every rabbit day oral 5mL Herba Medicaginis juice (2g/ day fresh leaf), 3 weeks, immunization time interval.

2.4.2 the mensuration of specific antibody IgG in immune serum

(1) sample treatment

Immunity gathers immune mouse and immune rabbit blood, 37 DEG C of coagulant 1h, 4 DEG C of left undisturbed overnight, the centrifugal 5min of 4000r/min on the 0th, 15,29,43,57,71,99 day, and collect serum ,-40 DEG C save backup.

(2) the alpha toxin proteantigen of expression of recombinant e. coli is prepared

BL21 (DE3) pET-30b-a bacterium liquid carries out SDS-PAGE electrophoresis after IPATG induction, through coomassie brilliant blue staining, cut alpha toxin albumen object band, electroelution spends the night, ultraviolet spectrophotometer measures eluted protein content (17mg/mL), detects by being used for ELISA as antigen after 1000 times of dilutions.

(3) ELISA detects

By the welch's fusiform bacillus alpha toxin protein of expression of recombinant e. coli with 1: 1000 dilution, get 100 μ L/ holes as antigen coated ELISA Sptting plate, 4 DEG C of bags are spent the night; Close with the PBS liquid 37 DEG C containing 5% skimmed milk and spend the night; Add Mus or the rabbit anteserum sample of 100 μ L/ hole 1: 100 dilutions, 37 DEG C of reaction 1h; Add HRP labelling sheep anti mouse or the goat anti-rabbit igg 100 μ L/ hole of 1: 3000 dilution, 37 DEG C of reaction 1h add OPD-H ₂o ₂substrate nitrite ion 100 μ L/ hole, 37 DEG C of lucifuge colour developing 5 ~ 10min, after adding 50 μ L/ hole stop buffers, enzyme mark analyzer measures the light absorption (OD in every hole at wavelength 490nm place ₄₉₀) value.

After continuous five oral immunity transgenic pA and pLA Herba Medicaginis, rabbit and mice all create the IgG antibody of anti-welch's fusiform bacillus alpha toxin protein higher level, as shown in Figures 1 and 2: within the 0th, 15,29,43,57,71,99 day, gather oral transgenic alfalfa and non-transgenic alfalfa immune group rabbit and mice serum sample 1: 100 respectively at immunity and dilute, using the welch's fusiform bacillus alpha toxin protein of expression of recombinant e. coli as antigen, elisa (ELISA) detects the IgG level in each group of sample.In rabbit serum sample, IgG antibody persistent levels raises, but rabbit first three exempt from ramp-up rate slowly (immunize rabbit grain), rear two exempt from ramp-up rate very fast (immune Herba Medicaginis juice).In mice serum sample, the horizontal rate of climb of IgG antibody comparatively steadily (equal immune Herba Medicaginis juice), continues to decline (see the 57th day) to some extent after 2 weeks.From induction IgG antibody level, transgenic alfalfa immune group is all apparently higher than non-transgenic alfalfa immune group, and immune pLA transgenic alfalfa group is all better than immune pA transgenic alfalfa group.

2.4.3 immune animal specific antibody IgA measures

(1) sample treatment

The process of fecal specimens: gather rabbit and stool in mice on the 0th, 15,29,43,57,71,99 day respectively at immunity, in units of immune group, every 0.1g feces adds 0.4mL feces lysate (containing 0.05mol/LEDTA-Na ₂pBS), be placed on agitator, vibration 4 ~ 5h, 4 DEG C of infiltrations are spent the night.The centrifugal 5min of 10000r/min, collect supernatant ,-20 DEG C of preservations are stand-by.

The collection of outer reproductive tract flushing liquor: gather rabbit and the outer reproductive tract washing liquid of mice on the the 0th, 15,29,43,57,71,99 day respectively at immunity, in units of immune group, repeatedly rinse rabbit and the outer reproductive tract of female mice with 200 μ LPBS, collect washing liquid ,-20 DEG C of preservations are stand-by.

The collection of nasal irrigation liquid: gather rabbit and mice nasal irrigation liquid on the 0th, 15,29,43,57,71,99 day respectively at immunity, in units of immune group, repeatedly rinse rabbit and mice nasal cavity respectively with 300 μ L and 100 μ L PBS, collect flushing liquor ,-20 DEG C of preservations are stand-by.

The collection of AK-Rinse: gather mice AK-Rinse on the 0th, 15,29,43,57,71,99 day respectively at immunity, in units of immune group, repeatedly rinse little rathole conjunctiva with 50 μ LPBS, collect flushing liquor ,-20 DEG C of preservations are stand-by.

The collection of intestinal mucus: immunity the 99th day each immune group randomly draws four mices, and under aseptic condition, eyeball gathers blood, and serum is used for alpha toxin antibody neutralization test; Dissect mice simultaneously, cut the small intestinal without feces, collect mucus, suitably dilute with PBS ,-20 DEG C of preservations.

(2) ELISA detects

Diluted by the welch's fusiform bacillus alpha toxin protein 1: 1000 of expression of recombinant e. coli, get 100 μ L/ holes as antigen coated 96 hole polystyrene micro plates, 4 DEG C of bags are spent the night; Close with the PBS liquid 37 DEG C containing 5% skimmed milk, spend the night; Add respectively handle well 100 μ L/ hole stool supernatant, AK-Rinse or nasal irrigation liquid, outer reproductive tract flushing liquor and mice intestinal mucus, 37 DEG C of reaction 1h; Add HRP labelling sheep anti mouse or the goat-anti rabbit IgA100 μ L/ hole of 1: 3000 dilution, 37 DEG C of reaction 1h; Add OPD-H ₂o ₂substrate nitrite ion 100 μ L/ hole, 37 DEG C of lucifuge colour developing 15min, after adding 100 μ L/ hole stop buffers, enzyme mark analyzer measures the light absorption (OD in every hole at wavelength 490nm place ₄₉₀) value.

1. anti alpha toxin specificity sIgA testing result in rabbit and stool in mice

As shown in accompanying drawing 3 and accompanying drawing 4, after continuous five immunity, rabbit and mice all create anti-A type welch's fusiform bacillus alpha toxin protein sIgA antibody, and in the fecal sample that after each immunity, 1 week (see the 15th, 29,43,71 and 99 day) gets, sIgA antibody horizontal continues to raise.Rabbit first three to exempt from the horizontal ramp-up rate of antibody sIgA slow equally, rear two to exempt from ramp-up rate very fast, and this is different from oral immunity mode relevant.It is also relatively steady that mice five exempts from the horizontal rate of climb of antibody sIgA, and antibody horizontal continues to decline (see the 57th day) equally to some extent after 2 weeks.From induction sIgA antibody horizontal, pLA transgenic alfalfa group is higher than pA group.

2. anti alpha toxin specificity sIgA testing result in the outer reproductive tract flushing liquor of rabbit and mice

As shown in accompanying drawing 5 and accompanying drawing 6, it is slow that rabbit sample exempts from the sIgA antibody horizontal rate of climb at first three, occurred declining in the 57th day; After the 4th booster immunization, sIgA antibody horizontal sharply rises, and this and oral way change relevant.Compare sIgA antibody horizontal in fecal specimens and want high, may be relevant with fecal specimens processing mode.In the outer reproductive tract flushing liquor sample of mice, the sIgA antibody horizontal rate of climb is relatively stable, namely the antibody of higher level is there is after first immunisation, after booster immunization, antibody horizontal continues to raise, but rear twice booster immunization ramp-up rate eases up, may be relevant with extending 1 week interval time; Mouse propagation road flushing liquor sIgA antibody horizontal is relatively lower than fecal sample.

3. anti alpha toxin specificity sIgA testing result in rabbit and mice nasal wash

As shown in accompanying drawing 7 and accompanying drawing 8, it is identical with outer reproductive tract flushing liquor with fecal specimens that sIgA antibody horizontal totally secretes trend, almost synchronous with Serological IgG level.Rabbit nasal cavity is comparatively large, and flushing liquor is muddy, and protein content is many, sIgA expression compare fecal specimens and outer reproductive tract flushing liquor sample higher.Although and mice nasal cavity is less, when collecting sample, part saliva can be mixed with, so sIgA detection level is also higher than outer reproductive tract flushing liquor.

4. anti alpha toxin specificity sIgA testing result in mice eye flushing liquor and intestinal mucus

As shown in Figure 9, sIgA Analysis of levels of antibody result and fecal specimens, outer reproductive tract flushing liquor are identical with nasal wash, but due to eye relative mice of sampling comparatively painful, sampling amount is few, so other sample antibody content are not very high relatively, but general trend is identical.

Immunity gathers mice intestinal mucus on the 99th day, 1: 5 dilution, with the welch's fusiform bacillus alpha toxin protein of expression of recombinant e. coli as antigen, compare with non-immune mouse intestinal mucus, elisa (ELISA) testing result is shown in accompanying drawing 10, and from induction sIgA antibody horizontal, immune pLA transgenic alfalfa group is higher than pA transgenic alfalfa group, oral non-transgenic alfalfa also can produce a small amount of antibody, illustrates that Alfalfa Polysaccharide has certain adjuvant effect.

2.5A type clostridieum welchii pathogenicity

2.5.1A type clostridieum welchii Morphological Identification

Getting-70 DEG C of A type clostridieum welchii strains of preserving lines on blood plate, anaerobism, 37 DEG C of overnight incubation.Single colony inoculation that picking zone of hemolysis is larger digests in soup in 5mL green meat liver gastric enzyme, adds 2mL sterile liquid paraffin, 37 DEG C of anaerobism, static gas wave refrigerator 24h, smear for microscopic examination, observes A type clostridieum welchii thalli morphology.

2.5.2A type clostridieum welchii pathogenecity improves test

If the virulence of A type clostridieum welchii is very weak, even without therapeutic, then need recovered by virulence and improve test: get A type clostridieum welchii lumbar injection 2 BALB/c mouse that 200 μ L activate, inject every other day once.If mice is still not dead after three times, get its liver under aseptic condition to rule on blood plate, 37 DEG C of anaerobism, static gas wave refrigerator spend the night, next day smear for microscopic examination, choosing single colony inoculation that form is correct, zone of hemolysis increases digests in soup in green meat liver gastric enzyme, 37 DEG C of anaerobism, left undisturbed overnight.Next day, smear for microscopic examination, repeated pathogenicity with 2 mices, until after 200 μ LA type clostridieum welchii alpha-toxin lumbar injections, and interior death during mice 24h.

2.5.3A type clostridieum welchii alpha-toxin pathogenicity

Being cultivated with 1: 10 by A type clostridieum welchii correct for form is inoculated in green meat liver gastric enzyme digestion soup, add 2mL sterile liquid paraffin, 37 DEG C of anaerobism, static gas wave refrigerator 7h, get alpha-toxin supernatant 10 μ L, 50 μ L and 200 μ L lumbar injection 3 BALB/c mouse respectively; Get alpha-toxin supernatant 50 μ L, 300 μ L and 500 μ L respectively ear vein inject 3 rabbits, at least observe 24h after injection, record and judge the virulence of A type clostridieum welchii alpha-toxin.

The pathogenicity of table 2-1A type clostridieum welchii alpha-toxin

Injected dose (mice) dead quantity death time injected dose (rabbit) the dead quantity death time

10 μ L (3) 00 50 μ L (2) 00

50 μ L (3) 2 16 ~ 42h 300 μ L (2) 2 15 ~ 38h

200 μ L (3) 38 ~ 12h 500 μ L (2) 26 ~ 11h

Inject in 3 non-immune mouses of 50 μ L and have 2 to occur diarrhoea, refusing to eat, in 24h dead one, in 48h dead one, also have one after injection 3h lethargy, refusal take food, be slow in action, breathe acceleration, slowly recover after about 12h, normally take food after 24h; 3 mices of injecting 200 μ L toxin are all dead in 12h, prove that toxin dose is bigger than normal; And 3 mices of injecting 10 μ L toxin are all normally taken food in 24h, without any abnormal response, illustrate that this dosage is on the low side.Equally, all there is diarrhoea in 2 rabbits of injecting 300 μ L, water is refused in refusing to eat, in 24h dead one, in 48h dead one; And 2 rabbits of injecting 50 μ L toxin all can normally be taken food in 24h, illustrate that using dosage is on the low side.

2.6 immune animals attack toxin test and serum antibody neutralization test

2.6.1 Immunization experimental animal

Lethal dose measures with animal: with immune group not immune BALB/c mouse of the same age or non-immune rabbit of the same age.

Counteracting toxic substances experimental animal: five exempt from after the immune mouse of 1 week, each immune group is divided at random six groups, each group four mices (male and female half and half), five groups are used for challenge test, and another group obtains a large amount of serum by eye blood sampling and is used for alpha-toxin antibody neutralization test; Five exempt from the rabbit of latter 1 week all for counteracting toxic substances.

2.6.2A type clostridieum welchii alpha-toxin challenge test

(1) mensuration of A type welch's fusiform bacillus alpha-endotoxin lethality amount

Get A type clostridieum welchii quiescent cultures alpha-toxin supernatant, by 15 μ L, 25 μ L, 50 μ L, 100 μ L, 150 μ L and 200 μ L, six various dose lumbar injections, 6 groups of white mice; 4 groups of rabbits are injected by 50 μ L, 100 μ L, 300 μ L and 500 μ L, tetra-various dose ear veins, at least 24h is observed after injection, judge the lethal dose of A type clostridieum welchii alpha-toxin: minimum lethal dose (the minimum lethal dose of non-immune mouse A type clostridieum welchii alpha-toxin, MLD) be 25 μ L, median lethal dose(LD 50) (LD ₅₀) be 50 μ L, absolute lethal dose (LD ₁₀₀) be 150 μ L; The MLD of non-immune rabbit A type clostridieum welchii alpha-toxin is 100 μ L, LD50 is 300 μ L, LD ₁₀₀be 500 μ L.

The determination experiment of table 2-2 alpha-toxin lethal dose

The dead quantity of dosage (μ L) mice quantity death toll amount dosage (μ L) rabbit quantity

15 3 0 50 2 0

25 4 1

50 4 2 100 3 1

100 4 3 300 4 2

150 4 4 500 4 4

200 3 3

(2) counteracting toxic substances infection experiment

After five immunity terminate, 1 week is the 99th day, carries out clostridieum welchii alpha-toxin challenge test to test I ~ III group and non-immune group experimental animal.20 mices in each experimental group are divided into five groups at random, respectively the alpha-toxin of lumbar injection 2MLD, 4MLD, 6MLD, 8MLD and 10MLD; In each experimental group 6 rabbit is divided into three groups at random, and the alpha-toxin of 2 injections of rabbit ear vein 3MLD, 5MLD and 7MLD in each experimental group, at least observes 24h, judges the immune protective effect of Oral transgene Herba Medicaginis according to result.Five exempt from mice after after immunity, 1 week (the 99th day) has adopted sample, and by table 2-3 grouping, often organize four mices, the alpha-toxin of lumbar injection different multiples MLD carries out challenge test respectively.

Table 2-3 clostridieum welchii alpha-toxin is to the challenge test of mice

Group 2MLD 4MLD 6MLD 8MLD 10MLD

PLA Herba Medicaginis immune group 00001

PA Herba Medicaginis immune group 00012

Non-transgenic alfalfa immune group 12344

Non-immune group 23444

Dead mouse result display after counteracting toxic substances: pA group can be resisted 6MLD completely and (is equivalent to LD ₁₀₀) attack of A type clostridieum welchii alpha-toxin, when 8MLD attacks protective rate be 75%, 10MLD when attacking protective rate be 50%; PLA immune group at least can resist the A type welch's fusiform bacillus alpha-toxin attacks of 8MLD, and when 10MLD attacks, protective rate is 75%; And non-immune group mice is all dead in 24h after 6MLD attacks, shows that transgenic alfalfa immune mouse can resist the strong virus attack of natural A type clostridieum welchii alpha-toxin, produce good immanoprotection action.

Table 2-4 clostridieum welchii is to the counteracting toxic substances infection experiment of rabbit

Group 3MLD 5MLD 7MLD

PLA Herba Medicaginis immune group 001

PA Herba Medicaginis immune group 012

Non-transgenic alfalfa immune group 122

Non-immune group 222

The A type clostridieum welchii alpha-toxin of rabbit 1 week ear vein injection different multiples MLD after five immunity, carries out challenge test.Rabbit death outcome (table 2-4) display after counteracting toxic substances: pA immune group at least can be resisted 3MLD and (is equivalent to LD ₅₀) attack of A type clostridieum welchii alpha-toxin, 5MLD (is equivalent to LD ₁₀₀) protective rate is that 50%, pLA group can be resisted 5MLD completely and (is equivalent to LD when attacking ₁₀₀) attack, when 7MLD attacks, protective rate is 50%, and all death in 24h after 5MLD attacks of non-transgenic alfalfa immune group and the non-immune group of negative control, can produce immanoprotection action after showing transgenic alfalfa immune rabbit.

2.6.3A type welch's fusiform bacillus alpha-toxin antibody neutralization test

A type clostridieum welchii virulent strain is inoculated in meat liver gastric enzyme digestion soup with 1: 10, and 37 DEG C of anaerobism, static gas wave refrigerator 7h, get culture fluid alpha-toxin supernatant for antibody neutralization test.

Table 2-5 mouse antibodies neutralization test

Dead mice (only) protective rate (%) of group

Equivalent physiological saline solution 50

Dilute 10 times of non-immune mouse serums 50

Dilute 10 times of non-transgenic alfalfa immune mouse serums 4 20

Dilute 10 times and turn pA Herba Medicaginis immune mouse serum 2 60

Dilute 10 times of pLA and turn Herba Medicaginis immune mouse serum 1 80

Get five exempt from after the turning pA Herba Medicaginis immune mouse serum, turn pLA Herba Medicaginis immune mouse serum of collection in 1 week, non-transgenic alfalfa immune mouse serum and non-immunonegative Mus serum, dilute 10 times with physiological saline solution, (be equivalent to LD with equivalent 6MLD respectively ₁₀₀) mixing of A type clostridieum welchii alpha-toxin, in contrast, 37 DEG C act on 30min to physiological saline solution.Often organize and get 150 μ L lumbar injection BALB/c mouse 5 respectively, at least observe 24h, record death and survival condition.Dead mouse result after attacking 24h (table 2-5) display: pA immune group serum dilute 10 times in be 60% with protective rate during 6MLDA type welch's fusiform bacillus alpha-toxin attacks; pLA immune group dilute serum protective rate is 80%; and non-immune group and normal saline group are after 6MLD attacks; all dead in mice 24h; show in transgenic alfalfa immune serum, to there is higher alpha toxin antibody; still can neutralize most of alpha toxin after diluting 10 times, serve immanoprotection action.

Claims (4)

1. a manufacture method for anti-clostridieum welchii disease Transgenic Plant Vaccines, is characterized in that: the method comprises:

(1) with containing the plasmid of coding clostridieum welchii alpha toxin or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain welch's fusiform bacillus alpha toxin gene, the welch's fusiform bacillus alpha toxin gene of acquisition and Ti-plasmids are carried out enzyme action respectively, reclaim genes of interest and plant expression vector fragment, connect and transformation of E. coli, enzyme action is carried out to restructuring Ti, PCR and order-checking qualification, use recombinant plasmid transformed agrobacterium tumefaciens, screening, PCR qualification obtains positive restructuring agrobacterium tumefaciens pA: be preserved in China typical culture collection center on June 2nd, 2010, preserving number is: CCTCC NO:M 2010130,

(2) alfalfa seed is through vernalization, sprouting, montage, obtain explant, explant is infected respectively with the agrobacterium tumefaciens pA of restructuring in step (1), by screening, subculture, take root, transplant, obtain transformed plant, Screening and Identification transgenic positive plant, extracts transgenic plant genome or albumen carries out Southern blot, Northern blot or Western blot identifies, proves that clostridieum welchii toxin is expressed in plant.

2. a kind of anti-clostridieum welchii disease Transgenic Plant Vaccines utilizing the method for claim 1 to make, it is characterized in that: described anti-clostridieum welchii disease Transgenic Plant Vaccines is direct oral vaccine, oral immunity produces secretory IgA, or by the proteantigen of expression of plants through extracting, being processed into oral or ejection preparation use.

3. a manufacture method for anti-clostridieum welchii disease Transgenic Plant Vaccines, is characterized in that: the method comprises:

(1) with containing the plasmid of mucosal adjuvant LTB (LT-B) or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain mucosal adjuvant LT-B gene, with containing the plasmid of coding clostridieum welchii alpha toxin or pathological material of disease for template, utilize PCR or RT-PCR to increase and obtain welch's fusiform bacillus alpha toxin gene, described mucosal adjuvant LT-B gene and described welch's fusiform bacillus alpha toxin gene are merged and obtains fusion gene, acquisition fusion gene and Ti-plasmids are carried out enzyme action respectively, reclaim genes of interest and plant expression vector fragment, connect and transformation of E. coli, enzyme action is carried out to restructuring Ti-plasmids, PCR and order-checking qualification, use recombinant plasmid transformed agrobacterium tumefaciens, screening, PCR qualification obtains positive restructuring agrobacterium tumefaciens pLA: be preserved in China typical culture collection center on June 2nd, 2010, preserving number is: CCTCC NO:M 2010131,

(2) alfalfa seed is through vernalization, sprouting, montage, obtain explant, explant is infected respectively with the agrobacterium tumefaciens pLA of restructuring in step (1), by screening, subculture, take root, transplant, obtain transformed plant, Screening and Identification transgenic positive plant, extracts transgenic plant genome or albumen carries out Southern blot, Northern blot or Western blot identifies, proves that clostridieum welchii toxin is expressed in plant.

4. a kind of anti-clostridieum welchii disease Transgenic Plant Vaccines utilizing claim 3 method to make, it is characterized in that: described anti-clostridieum welchii disease Transgenic Plant Vaccines is for directly oral, oral immunity produces secretory IgA, or by the proteantigen of expression of plants through extracting, being processed into oral or ejection preparation use.