CN103865933B - The application of a kind of WAP gene in Drosophila transgenic - Google Patents
The application of a kind of WAP gene in Drosophila transgenic Download PDFInfo
- Publication number
- CN103865933B CN103865933B CN201410075171.0A CN201410075171A CN103865933B CN 103865933 B CN103865933 B CN 103865933B CN 201410075171 A CN201410075171 A CN 201410075171A CN 103865933 B CN103865933 B CN 103865933B
- Authority
- CN
- China
- Prior art keywords
- wap
- fly
- gene
- transgenic
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The present invention relates to the application of a kind of WAP gene in Drosophila transgenic, the present invention passes through gene constructed for the WAP in Chinese prawn in fruit bat, and WAP gene is expressed in fruit bat, and transgenic fly prepared by the present invention has antibacterial effect.
Description
Technical field
The present invention relates to the application of a kind of WAP gene in Drosophila transgenic.
Background technology
Even abuse along with microbiotic is widely used, antibiotic remains and bacterial resistance sex chromosome mosaicism more and more receive global concern, and research and development novel antibacterial medicine carrys out alternative conventional antibiotic and also to become international brainstorm subject.
Antibacterial peptide is the micromolecule polypeptide with strong anti-microbial effect produced in organism, when resisting the pathogenic bacteria invasions such as bacterium, virus, fungi, there is very important defense reaction, be considered to the novel anti-infection medicine being difficult to cause microorganism resistance, treatment, preventing cancer and antiviral, anti-infective etc. in have a good application prospect, become the focus (SplithandNeuntorf, 2011) of new antimicrobial agent research and development.Antibacterial peptide product is the Substitutes For Antibiotic being in world lead level, may be used for field (Fernebro, 2011 such as medicine company, foodstuffs industry, livestock industry, culture fishery, daily-use chemical industry industry; Mar ó tietal., 2011).Therefore, antimicrobial peptide medicaments is researched and developed significant to the development facilitated economic and social.
In recent years, rapidly, annual growth reaches 19%(Liang Yuan army and Liu Keliang for global protein, polypeptide drug market development, and 2007).The U.S. once the world the first report antibacterial peptide---cecropin has researched and developed the medicine into feed anticorrosion agent and wound washing composition, is called as " omnipotent peptide ".Japan has the patent using lectin as Therapeutic cancer preparation to declare.China's Scientific Research Workers has also carried out much useful work in the research and development of anti-mattress peptide medicine.At the beginning of 1999, the unit consolidation applications such as Agricultural University Of South China high-tech achievement hatching project " test manufacture of Antibacterial Peptide-sterilizing and anti-virus new drug ", the Antibacterial Peptide of research and development is to Gram-negative and positive bacteria, drug tolerant bacteria all has germicidal action, can suppress copying of hepatitis B virus DNA, energy selective killing tumour cell, can be further purified as monomer makes new drug, as the oral pharmaceutical (yellow nature, 1999) for the treatment of patience bacteriological infection.2010, Kunming Institute of Zoology, Chinese Academy of Sciences and Suzhou brilliance industry (group) company limited developed jointly antimicrobial peptide medicaments (" Chinese Medicine biotechnology ", 2010).The scientific research institutions such as Life Science and Technology College, Xinjiang Univ. utilize genetic engineering means to obtain high reactivity Sinkiang domestic silkworm antibiotic peptide, there is the effect suppressing multiple malignant bacteria and dental decayed tooth bacterium, also to the growth of relevant tumour cell, there is obvious restraining effect, can research and develop as sterilization, antibacterial, anti-dental caries, antineoplastic antimicrobial peptide medicaments (http://www.tianshannet.com, 2010)." antibacterial peptide research and development, construction project " project is newly gone up in the investment 8 hundred million in 2010 of the East Sea, Zhejiang Bioceuticals Inc.; main products is the antibacterials " antibacterial peptide Thanatin and mutant thereof " of substitute antibiotics of new generation; 1 ton, the former powder of antibacterial peptide is produced in plan per year, can save the life of 2,000,000 critically ill patients (using the antibiotic ineffective persons such as penicillin, Streptomycin sulphate, terramycin, duomycin) according to a preliminary estimate every year.Therefore, antimicrobial peptide medicaments is difficult to estimate in the social effect of saving in life, guarantee human health and economic worth.
Although antibacterial peptide shows huge new drug development potentiality, but it is not most antibacterial peptide also also exists a lot of problem as drug manufacture, such as easily high by the recombinant protein indissoluble of protease hydrolysis, organism intensive amount too low direct separation and purification difficulty, prokaryotic expression from biological tissue, output and activity without protease inhibiting activity.At present, the antimicrobial peptide medicaments succeeded in developing is also considerably less.
The patent No. is 20051004458888 patent discloses in Chinese prawn and cloned antibacterial peptide gene (being abbreviated as WAP) (JiaYP containing single whey acidic protein structure domain, 2008), and utilize prokaryotic expression WAP antibacterial peptide recombinant protein, it exists with insoluble inclusion bodies, through sex change, renaturation, obtain pure WAP antibacterial peptide recombinant protein, this WAP antibacterial peptide of vitro detection has the difunctional of broad spectrum antibiotic activity and protease inhibiting activity, shows that WAP antibacterial peptide has good medicament research and development prospect.
Research and development stable performance, active by force, not only antibacterial the but also bifunctional WAP antibacterial peptide newtype drug with protease inhibiting activity be exactly point of penetration of the present invention.
Summary of the invention
The invention provides the application of a kind of WAP gene in Drosophila transgenic, it utilizes transgenic technology, and the wap gene of Chinese prawn is proceeded to drosophila gene group, obtains high expression level, genetic stability, has the WAP transgenic fly strain of antibacterial; Express the transgenic fly of WAP feed the poultry such as chicken, fish and fish and water biological, there is the significantly disease-resistant medicinal effect with weightening finish.
Concrete technical scheme of the present invention is as follows:
(1) structure of transgenic fly: according to ORF two terminal sequence of WAP gene, designs a pair upstream and downstream primer Wap-f and Wap-r, and introduces respectively at 5 ' end of upstream and downstream primer
kpni with
xbai restriction enzyme site, primer sequence is as follows:
Wap-f：5’---GG
GGTACC ATGGTGAACATCAAGGAAGTTC---3’（SEQIDNO：1）
Wap-r：5’---GCTCT
AGATCA TTTTCCGTAGGGAGATCCCA---3’（SEQIDNO：2）
With Hemocytes In Chinese Shrimp Penaeus Chinensis cDNA for template, PCR fishes and gets WAP fragment, utilizes
kpni He
xbai two restriction enzyme sites, are connected with pUAST plasmid orientation, build carrier for expression of eukaryon wap/pUAST, WAP fragment sequence (SEQIDNO:1) (SEQIDNO:2) through inserting in PCR checking and this plasmid of sequence verification correctly after, by wap/pUAST and
Δ 2-3helper plasmid together microinjection enters the wild-type drosophila melanogaster W of the supercilious look
1118embryo in,
Δ 2-3on helper plasmid transposon P element assistance under, WAP gene by random integration in the genome of drosophila embryos pole cell, pole cell develops into sexual gland in the future, and the G0 generation of injecting successful fetal development becomes the genetically modified gamete of fly meeting generating strap wap, when G0 becomes fly and W
1118during drosophila hybrid, there will be wap transgenosis in hybrid generation (G1) and successfully see red fruit bat, because wap/pUAST there is mini-white marker gene, make drosophila compound eye present redness, according to this examination, just obtain the transgenic fly strain of seeing red; Utilize PCR, from transgenic fly strain genome, be checked through WAP gene fragment, prove that WAP transgenic fly successfully constructs;
Described wap gene is the antibacterial peptide gene that Chinese prawn contains single whey acidic protein structure domain, its nucleotide sequence following (SEQIDNO:3):
ATGGTGAACATCAAGGAAGTTCTGATCGTG30
TCCGTGTTGGTGGCCGCGGTGGCTGTTTCT60
CCCGCCGATGCTGTTCCAACGAGACACGCT90
AGGCCCCGTCCTCAGCCCAGGCCGAGGCCA120
GGGACGTGTCCGGACACGAGCGACATCGTC150
TCCATCTGCGTCGTGACGGAACGCAACTGC180
TTCTCGGACGGCGAGTGCGGAGCCGGCCAG210
AAGTGCTGTCCGATTGGCTGCGGGAGAGAG240
TGCCTGGCTGTGGGATCTCCCTACGGAAAATGA273
Its aminoacid sequence following (SEQIDNO:4):
MVNIKEVLIVSVLVAAVAVSPADAVPTRHA30
RPRPQPRPRPGTCPDTSDIVSICVVTERNC60
FSDGECGAGQKCCPIGCGRECLAVGSPYGK90
Described
kpnthe nucleotides sequence of I is classified as
gGTACC;
Described
xbathe nucleotides sequence of I is classified as
tCTAGA.
(2) assignment of genes gene mapping of wap in transgenic fly: from transgenic fly strain, selects the male fly of WAP transgenosis of blood-shot eye illness, respectively with equilibrium system (w-/w-; Sco/Cyo; Sb/Tb) through two-wheeled hybridization, according to the separation phenomenon of filial generation fruit bat phenotype, located wap in No. I karyomit(e), No. II karyomit(e) or No. II and No. III chromosomal 5 WAP transgenic fly strains.
(3) expression of WAP in transgenic fly body: the WAP transgenic fly of having good positioning and Gal4 fruit bat are hybridized, obtains the transgenic fly of expressing wap gene.
The expression of WAP in transgenic fly body is by the regulation and control of Gal4-UAS double element system, because different Gal4 strains provides the Gal4 expressed at different tissues, and the wap gene in WAP transgenic fly is positioned at the downstream (UAS-wap) of UAS, during different from actin-Gal4, He-Gal4, ppl-Gal4, elav-Gal4 when WAP transgenic strain Gal4 incross, filial generation is provided with Gal4/UAS-wap double element system, Gal4 can be combined with UAS, and the wap gene the ordering about UAS downstream after hybridization whole body in generation, hemocyte, fatty body or neural system is expressed; And WAP transgenic strain, different Gal4 strains and wild-type W
1118the filial generation of fruit bat, does not possess Gal4/UAS-wap double element system, does not almost have the expression of wap gene.QPCR detected result also demonstrate that this point: the filial generation possessing Gal4/UAS-wap double element system, the expression amount of its wap gene is higher than the contrast more than 100 times not expressing wap gene, and the expression level of wap gene in the filial generation of different transgenic strain has high, normal, basic difference.
The transgenic fly that the present invention is prepared into can be prepared into transgenic fly medicine, and it has significant anti-microbial effect.
The WAP transgenic fly that the present invention builds, by regulation and control and the hybrid experiment of Gal4/UAS-wap double element system, achieve the high expression level of WAP antibacterial peptide in fruit bat particular organization, not only overcome the indissoluble of prokaryotic expression WAP recombinant protein, lack the shortcoming such as posttranslational modification and active reduction, and avoid the trouble of prokaryotic expression WAP albumen aftertreatment, by means of only hybridization, just can using WAP as fixed point administration endo-medicine introduce in the particular organization of transgenic fly, improve bacteria resistance function and the viability of the fruit bat of expressing WAP, simultaneously, WAP expression amount in transgenic fly is high, Direct-fed poultry and fish have disease-resistant gaining effect, to the function causing scorching fish and also have external application sterilization, three, by the amount reproduction of fruit bat, easily results WAP antimicrobial peptide medicaments product, is beneficial to suitability for industrialized production.
WAP transgenic fly disclosed by the invention is a kind ofly convenient to WAP antimicrobial peptide medicaments product that is for oral administration and external application, have produce WAP antimicrobial peptide medicaments simple and convenient, easy storage, environmental protection, be convenient to repetition, advantage that cost is low, be suitable for poultry and culture fishery.
Accompanying drawing explanation
Accompanying drawing 1 is the result that the carrier for expression of eukaryon wap/pUAST built verifies through PCR.Use WAP special primer: Wap-f and Wap-r carries out PCR detection to 5 wap/pUAST plasmids, obtain and with Hemocytes In Chinese Shrimp Penaeus Chinensis cDNA be template PCR amplifications positive findings WAP fragment of a size.
Accompanying drawing 2 is carrier for expression of eukaryon wap/pUAST results through sequence verification of structure.With the comparison of wap gene order, find that the wap fragment sequence inserted in wap/pUAST is entirely true.
Accompanying drawing 3 is PCR detected results of WAP transgenic fly.Extract WAP transgenic fly and W
1118the genomic dna of wild-type drosophila melanogaster, use WAP special primer respectively: Wap-f and Wap-r carries out PCR detection to these genomic dnas, to amplify from WAP transgenic fly genome and be the equirotal fragment of template amplification WAP with wap/pUAST positive plasmid, and from W
1118wap target fragment is not amplified in the genome of wild-type drosophila melanogaster.Prove that Chinese prawn wap gene is successfully inserted in the genome of WAP transgenic fly.
Accompanying drawing 4 is wap assignment of genes gene mapping schemas in transgenic fly.First, from transgenic fly tx5, t6, t10, t32, t78 strain of carrying wap, the male fly of a blood-shot eye illness is respectively chosen, respectively with w-/w-; Sco/Cyo; Sb/Tb equilibrium system virgin fly hybridizes, and observes the phenotype of F1 generation.If there is the sexual segregation of blood-shot eye illness, then can locate wap gene on I karyomit(e), if asexuality is separated, namely in male and female individuality, all have blood-shot eye illness to occur, infer that wap may be inserted on II or III karyomit(e).Then, being inserted in I chromosomal F1 generation fruit bat from eliminating wap, selecting the male fly of blood-shot eye illness with the sub-genetic stability of balance, as stuck up wing, short stubble, blood-shot eye illness (Cyo; Sb, red) or stick up wing, short pupa, bristle clusters, sees red (Cyo; Tb, red) male fly, then with equilibrium system (w-/w-; Sco/Cyo; Sb/Tb) virgin fly carries out second and takes turns hybridization, observes F2 and represents type.If appearance is seen red, few hair sticks up wing (+/+; Sco/Cyo) phenotype, then infer that wap goal gene is inserted on No. III karyomit(e); If appearance blood-shot eye illness, short stubble, short pupa, bristle are clustered (+/+; Sb/Tb) phenotype, then deducibility wap goal gene is inserted on No. II karyomit(e).
Accompanying drawing 5 is that QPCR detects the expression of results of WAP in transgenic fly.Get 5 transgenic fly strains: the male fly of t6, t78, tx5, t10, t323, hybridize with Actin-Gal4 virgin fly respectively and make experimental group, be labeled as t6xrj63, t78xrj63, tx5xrj63, t10xrj63, t32xrj63, Parent respectively with W
1118drosophila hybrid compares group, and male parent contrasting marking is t6, t78, tx5, t10, t32, and maternal contrast is not marked.The total serum IgE reverse transcription cDNA extracting experimental group, male parent and maternal control group hybridization first filial generation sample is template, does internal reference, carry out QPCR detection with Wap-f and Wap-r primer with house-keeping gene actin.Found that, on rna level, maternal control group offspring expresses without WAP, male parent control group offspring has few WAP to express, and experimental group offspring all has WAP high expression level, and WAP expression amount is up to more than 100 times of male parent control group offspring, in the filial generation of 5 transgenic strains, what WAP expression amount was the highest is from t6, is secondly t78, is next t10, t32 and tx5 successively.
Accompanying drawing 6 is detected results of WAP transgenic fly antibacterial in high-concentration bacterial environment.X-coordinate is the emergence period of 1-12 days, and ordinate zou is mortality ratio.In emergence period, the mortality ratio of the transgenic fly of expressing WAP and the Parent of not expressing WAP contrast fruit bat increases all in time and improves in corresponding, and the mortality ratio of control group fruit bat is higher than the mortality ratio of experimental group more than 2 times.Illustrate: on the one hand, along with the increase of incubation time, in substratum, the concentration of bacterium constantly increases, and accelerates the death of fruit bat, improves the mortality ratio of fruit bat; On the other hand, compare photograph, the expression of WAP improves the antibacterial ability of transgenic fly, reduces their mortality ratio (see the curve of three in figure), also shows, in the environment of high bacteria concentration, WAP transgenic fly compares according to having higher immunizing power.
Accompanying drawing 7 is detected results of the lower WAP transgenic fly antibacterial of bacterium thorn.Choose offspring's hero fly C ♂ and each 300 of female fly C ♀ of the male fly A ♂ and female fly A ♀ of offspring of WAP Transgenic male fruit bat and He-Gal4 virgin fly hybrid experiment group, the male fly B ♂ and female fly B ♀ of offspring of male parent control group, maternal control group, with dip intestinal bacteria (
escherichiacoli), staphylococcus aureus (
staphylococcusaureus) (bacteria concentration is 1.13 × 10 to mixed bacterium solution
9cFU/mL) 1mL asepsis injector syringe needle stimulates, every fruit bat 1 pin, and do the same fruit bat that stimulates and normal inirritative fruit bat contrasts to dip LB, statistics stimulates fruit bat survival numeral system in latter 6 days to make curve.
Embodiment
The present invention is further illustrated below.
(1) structure of WAP transgenic fly: according to ORF two terminal sequence of Chinese prawn containing the antibacterial peptide gene WAP of single whey acidic protein structure domain, design a pair upstream and downstream primer Wap-f and Wap-r, and introduce respectively at 5 ' end of upstream and downstream primer
kpni(GGTACC) with
xbai(AGATCA) restriction enzyme site, primer sequence is as follows:
Wap-f:5’---GG
GGTACCATGGTGAACATCAAGGAAGTTC---3’（SEQIDNO：1）
Wap-r:5’---GCTCT
AGATCAT
TTTCCGTAGGGAGATCCCA---3’（SEQIDNO：2）
With Hemocytes In Chinese Shrimp Penaeus Chinensis cDNA for template, PCR fishes and gets WAP fragment, utilizes
kpni He
xbai two restriction enzyme sites, are connected with pUAST plasmid orientation, build carrier for expression of eukaryon wap/pUAST, the WAP fragment sequence inserted in PCR checking and this plasmid of sequence verification correctly (see accompanying drawing 1,2), by wap/pUAST and
Δ 2-3helper plasmid is injected into the wild-type drosophila melanogaster W of the supercilious look together
1118486 embryos in,
Δ 2-3on helper plasmid transposon P element assistance under, wap gene is by random integration in the genome of drosophila embryos, and the successful embryo of transgenosis will develop into the one-tenth fly of blood-shot eye illness, through examination, obtain 10 blood-shot eye illness transgenic fly strains (see table 1).Utilize PCR, from transgenic fly strain genome, be checked through WAP gene fragment (see accompanying drawing 3), prove that WAP transgenic fly successfully constructs.
Table 1 transgenic fly statistics
| Embryonal vaccination | Larvae | Sprout wings into fly | Be hybridized to fly | Transgenic fly | Hatching rate | Eclosion rate | Transformation efficiency |
| 486 | 309 | 249 | 249 | 10 | 63.6% | 80.6% | 2.0% |
Table 1 builds WAP transgenic fly statistics.With wap/pUAST recombinant plasmid with
Δ 2-3helper plasmid mixed solution, injection wild-type drosophila melanogaster W
1118embryo 486, be injected embryo hatching go out 309 larvas, hatching rate is 63.6%, and these larvas sprout wings and G0 generation and become fly 249, and eclosion rate is 80.6%.Become fly male female with 4 by 2 G0 generation; 2 is female male with 2; 1 is male female with 2; The 1 female ratio with 1 hero, with the wild-type W of the opposite sex
1118fruit bat hybridizes, and obtains 127 cross combinations.In 127 cross combination, 100 combinations are had to create G1 hybrid generation, wherein find the WAP transgenic fly of seeing red in 5 G1 generations of combining, these 5 are combined the G0 fruit bat respectively having 2 to be fertilized, the transgenic fly strain formed is equivalent to 10, compared with embryonal vaccination number, and transgenosis ratio about 2.0%, by the order of cross combination numbering, that names these 5 to have a WAP transgenic fly is combined as tx5, t6, t10, t32, t78.
(2) assignment of genes gene mapping of WAP transgenic fly: from the G1 generation of tx5, t6, t10, t32, t78, selects the male fly of WAP transgenosis of single blood-shot eye illness, respectively with equilibrium system (w-/w-; Sco/Cyo; Sb/Tb) hybridize through two-wheeled, according to the separation phenomenon of filial generation fruit bat phenotype, in the G1 generation of learning t32 and t78, there is the wap assignment of genes gene mapping in the G1 generation of No. I chromosomal transgenic fly strain, t6, have the wap assignment of genes gene mapping to have the wap assignment of genes gene mapping respectively No. II, No. III chromosomal two transgenic fly strains in the G1 generation of No. II chromosomal transgenic fly strain, tx5 and t10.Positioning flow is shown in accompanying drawing 4.
(3) expression of WAP gene in transgenic fly body: the expression of wap gene in transgenic fly body is by the regulation and control of Gal4-UAS double element system, because different Gal4 strains provides the Gal4 expressed at different tissues, and the wap gene in WAP transgenic fly is positioned at the downstream (UAS-wap) of UAS, as WAP transgenic strain and actin-Gal4, He-Gal4, ppl-Gal4, during elav-Gal4 incross, filial generation is provided with Gal4-UAS double element system, Gal4 can be combined with UAS, drive the whole body of wap gene (UAS-wap) fruit bat in UAS downstream, hemocyte, fatty body or neural system are expressed, QPCR detects discovery, the expression amount of wap is higher than contrast more than 100 times, and at WAP transgenic fly and wild-type W
1118fruit bat or Gal4 fruit bat and wild-type W
1118in the filial generation contrast of fruit bat, because Gal4-UAS double element system is incomplete, cannot start the expression of wap, the expression amount of wap almost can't detect, and wap has high, normal, basic different at different WAP transgenic strain and the expression level in the filial generation of Gal4.See accompanying drawing 5.
(4) WAP transgenic fly antibacterial detects: hybridize WAP Transgenic male fruit bat and Gal4 virgin fly as experimental group, WAP transgenic fly and W
1118fruit bat, Gal4 fruit bat and W
1118the cross combination of fruit bat is Parent control group.
The first, experimental group and control group cultivate simultaneously containing intestinal bacteria (
escherichiacoli), staphylococcus aureus (
staphylococcusaureus), micrococcus luteus (
micrococcusluteus) (bacteria concentration is 1.2 × 10 to three kinds of mixed bacterium
7cFU/mL), on substratum, the offspring adding up them becomes the change of fly mortality ratio in emergence number of days.Result shows, and control group filial generation becomes the mortality ratio of fly up to 2 times of experimental group, illustrates in the environment of high bacteria concentration, and WAP transgenic fly compares according to having higher antimicrbial power (see accompanying drawing 6)
The second, choose experimental group and control group hybridization first filial generation male and female and become each 300 of fly, respectively with dip intestinal bacteria (
escherichiacoli), streptococcus aureus (
staphylococcusaureus) (bacteria concentration is 1.13 × 10 to mixed bacterium solution
9cFU/mL) 1mL asepsis injector syringe needle stimulates, every fly 1 pin, and contrast with the fruit bat and normal inirritative fruit bat that dip LB stimulation, statistics stimulates the survival number of fruit bat in latter 6 days.Result shows: the fruit bat survival number that normally inirritative and LB stimulates changes little, and compare photograph, bacterium thorn makes survival fruit bat obviously reduce, and the survival number of bacterium thorn experimental group fruit bat more than control group 2-7 doubly (see accompanying drawing 7).After bacterium stings 7 days, substantially all in the dust, the survival of experimental group fruit bat can reach 21 days to control group fruit bat, long and control group three times.The content of molds in the experimental group fruit bat body of surviving is detected: what bacterium was stung is every fruit bat 115-130 bacterium, and every fruit bat that LB stimulates has 0-3, normal nothing by colony counting method.Illustrate that bacterium stabs in fruit bat body and introduce a large amount of bacteriums, the fruit bat of expressing WAP can have better antibacterial ability than the control group fruit bat of not expressing WAP, causes their mortality ratio low long with the survival time.
(5) medicinal function of WAP transgenic fly detects: be added in feed by WAP transgenic fly by different amounts, raise housefly, loach, chicken, raise certain number of days, investigation feeding animals body weight increases situation and sickness rate, mortality ratio, to determine its effect.
Select housefly third-instar larvae and become each 100 of fly, respectively with dip intestinal bacteria (
escherichiacoli), streptococcus aureus (
staphylococcusaureus) (bacteria concentration is 1.13 × 10 to mixed bacterium solution
9cFU/mL) 1mL asepsis injector syringe needle stimulates, every fly 1 pin, they are divided into two groups: one group of feed conventional feed and water for contrast, another group is experimental group, is mixed with the WAP antibacterial peptide crude product (2000mg/kg) extracted from WAP transgenic fly in its feed and water.Prepared by WAP antibacterial peptide crude product: by expressing the fruit bat of WAP, pulverize with the ammonium acetate buffer of 0.5mol/L, and by 1:5(W/V) than when being immersed in that in this damping fluid, 4-5 is little, boil 10 minutes, 10000 leave the heart 10 minutes, collect supernatant, powder is made ,-20 DEG C of preservations with Freeze Drying Equipment.Survey the mortality ratio after feeding 3 days.Compare control group, experimental group microbiological contamination Musca domestica larva with become the mortality ratio of fly to reduce 17% and 21%, become fly prolonged survival period 12 days.Larvae pupation rate improves 2 times.In table 2.
Select the loach 300 of heavy 11 ± 1g, use 1mL asepsis injector, to loach subdermal muscle inject 100 μ L intestinal bacteria (
escherichiacoli), staphylococcus aureus (
staphylococcusaureus), subtilis (
bacillussubtilis) (bacteria concentration is 1.13 × 10 to solution
9cFU/mL), being divided into three groups: one group of conventional fish meal of feeding by there being 300 of obvious inflammation loach is contrast 1, one group of fish meal mixing penbritin (100mg/kg) of feeding is contrast 2, it is experimental group that another group feeds the fish meal mixing WAP fruit bat (3000mg/kg), raises 20 days.Compare according to 1, the mortality ratio of experimental group loach declines 22%, compares according to 2, inflammation cure time early 2 days.In addition, be control group with the normal loach of conventional fish meal of feeding, and the normal loach of mixing WAP fruit bat (3000mg/kg) fish meal of feeding is experimental group, compare photograph, experimental group loach rate of body weight gain improves 27%, and mortality ratio declines 7%, and vitality strengthens (see table 3).
Table 3WAP transgenic fly detects table to the medicinal function of loach
Select 300 chick (103 ± 1g), average mark 3 groups, often group provides different feeds: basal feed is control group 1, basal feed+penbritin (50mg/kg) is control group 2, basal feed+WAP fruit bat (2000mg/kg) is experimental group, raises 20 days, weigh under similarity condition.As a result, the rate of body weight gain of experimental group, surviving rate are compared with control group 1, high 39.8% and 5%, the ability that control group 2 and experimental group improve survivability of chicks is similar, but gaining ability is not obvious.In table 4.
Table 4WAP transgenic fly detects table to the medicinal function of chick
The present invention passes through RT-PCR technology, clone Chinese prawn wap gene, utilize transgenic technology, construct WAP transgenic fly, pass through drosophila hybrid, obtain 5 WAP transgenic fly strains that the wap assignment of genes gene mapping is clear and definite, by the driving of Gal4, wap gene high expression, the bacterium of transgenic fly to inside and outside of high expression level WAP has strong inhibit feature, namely the survival ability of himself can be improved, can also as medical feed additive, raise poultry and aquatic organism are had to the function of antibacterial, anti-inflammatory, weightening finish.Illustrate that WAP has the feature of novel antimicrobial peptide medicine.
Obviously, WAP transgenic fly can be introduced WAP in fruit bat body as the endo-medicine of fixed point administration by hybridization, improve bacteria resistance function and the viability of fruit bat self, simultaneously, by the amount reproduction of fruit bat, the WAP antimicrobial peptide medicaments product that easily results are a large amount of, as fodder additives external application, the bacterium of environment can be suppressed, also there is the medicinal function improving biological antibiotic, anti-inflammatory, weightening finish.
Therefore, the WAP transgenic fly that the present invention builds, can by the regulation and control of Gal4-UAS double element system and drosophila hybrid experiment, realize the high expression level of WAP antibacterial peptide in fruit bat particular organization, namely the shortcoming that the WAP recombinant protein indissoluble of prokaryotic expression, shortage posttranslational modification and activity are low is overcome, turn avoid the trouble of the WAP albumen aftertreatment of prokaryotic expression, as antimicrobial peptide medicaments product, have and produce simple and convenient, easy storage, environmental protection, be convenient to the advantage of repetition, be applicable to fruit bat, poultry and aquatic organism aquaculture and use.
<110> University Of Ji'nan
The <120> application of WAP gene in Drosophila transgenic
<160>2
<210>1
<211>30
<212>DNA
<213> artificial sequence
<220>
<221>misc_feature
<222>(1)..(30)
<223> primer
<400>1
GGGGTACCATGGTGAACATCAAGGAAGTTC30
<210>2
<211>31
<212>DNA
<213> artificial sequence
<220>
<221>misc_feature
<222>(1)..(31)
<223> primer
<400>2
GCTCTAGATCATTTTCCGTAGGGAGATCCCA31
<210>3
<211>273
<212>DNA
The biological name in this sequence of <213> source----Chinese prawn (
fenneropenaeuschinensis)
<220>
The antibacterial peptide of <221> single whey acidic protein structure domain
<400>3
ATGGTGAACATCAAGGAAGTTCTGATCGTG30
TCCGTGTTGGTGGCCGCGGTGGCTGTTTCT60
CCCGCCGATGCTGTTCCAACGAGACACGCT90
AGGCCCCGTCCTCAGCCCAGGCCGAGGCCA120
GGGACGTGTCCGGACACGAGCGACATCGTC150
TCCATCTGCGTCGTGACGGAACGCAACTGC180
TTCTCGGACGGCGAGTGCGGAGCCGGCCAG210
AAGTGCTGTCCGATTGGCTGCGGGAGAGAG240
TGCCTGGCTGTGGGATCTCCCTACGGAAAATGA273
<210>4
<211>90
<212> amino acid
The biological name in this sequence of <213> source----Chinese prawn (
fenneropenaeuschinensis)
<220>
The antibacterial peptide of <221> single whey acidic protein structure domain
<400>4
MVNIKEVLIVSVLVAAVAVSPADAVPTRHA30
RPRPQPRPRPGTCPDTSDIVSICVVTERNC60
FSDGECGAGQKCCPIGCGRECLAVGSPYGK90
Claims (2)
1. the application of WAP gene in Drosophila transgenic, is characterized in that, comprise the following steps:
The structure of transgenic fly: according to ORF two terminal sequence of WAP gene, designs a pair upstream and downstream primer Wap-f and Wap-r, and introduces kpnI and xbaI restriction enzyme site respectively at 5 ' end of upstream and downstream primer, and primer sequence is as follows:
Wap-f：5’---GGGGTACCATGGTGAACATCAAGGAAGTTC---3’
Wap-r：5’--GCTCTAGATCATTTTCCGTAGGGAGATCCCA---3’
With Hemocytes In Chinese Shrimp Penaeus Chinensis cDNA for template, PCR fishes and gets WAP fragment, utilize kpn I and xba I two restriction enzyme sites, be connected with pUAST plasmid orientation, build carrier for expression of eukaryon wap/pUAST, after WAP fragment sequence through inserting in PCR checking and this plasmid of sequence verification is correct, wap/pUAST microinjection together with Δ 2-3 helper plasmid is entered the wild-type drosophila melanogaster W of the supercilious look
1118embryo in, on Δ 2-3 helper plasmid transposon P element assistance under, WAP gene is by random integration in the genome of drosophila embryos pole cell, and pole cell in the future develops into sexual gland, in the G0 generation of injecting successful fetal development, becomes the genetically modified gamete of fly meeting generating strap wap, when G0 becomes fly and W
1118during drosophila hybrid, there will be wap transgenosis in hybrid generation and successfully see red fruit bat, because wap/pUAST there is mini-white marker gene, make drosophila compound eye present redness, according to this examination, just obtain the transgenic fly strain of seeing red; Utilize PCR, from transgenic fly strain genome, be checked through WAP gene fragment, prove that WAP transgenic fly successfully constructs;
Described WAP gene is the antibacterial peptide gene that Chinese prawn contains single whey acidic protein structure domain; Its nucleotide sequence is as shown in SEQIDNO:3; Its aminoacid sequence is as shown in SEQIDNO:4; The nucleotides sequence of described kpnI is classified as GGTACC; The nucleotides sequence of described xbaI is classified as TCTAGA.
2. the application of transgenic fly in preparation antibacterials of the wap genetic expression described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410075171.0A CN103865933B (en) | 2013-11-30 | 2014-03-04 | The application of a kind of WAP gene in Drosophila transgenic |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310612373 | 2013-11-30 | ||
| CN201310612373X | 2013-11-30 | ||
| CN201310612373.X | 2013-11-30 | ||
| CN201410075171.0A CN103865933B (en) | 2013-11-30 | 2014-03-04 | The application of a kind of WAP gene in Drosophila transgenic |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103865933A CN103865933A (en) | 2014-06-18 |
| CN103865933B true CN103865933B (en) | 2016-03-23 |
Family
ID=50904942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410075171.0A Active CN103865933B (en) | 2013-11-30 | 2014-03-04 | The application of a kind of WAP gene in Drosophila transgenic |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103865933B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104304185A (en) * | 2014-09-18 | 2015-01-28 | 中国医科大学 | Construction and screening of drosophila biological model |
| CN107287217B (en) * | 2017-06-30 | 2019-11-08 | 浙江大学 | CvBV29-1 gene is reducing drosophila immunity and is preparing the application in immunocompromised type transgenic drosophila model |
| CN108184770B (en) * | 2018-01-19 | 2020-11-03 | 华北理工大学 | Drosophila melanogaster RasV12Method for establishing Snail tumor migration model |
| CN108849765B (en) * | 2018-07-27 | 2021-07-02 | 江苏省农业科学院 | Microinjection and artificial hatching method for rice planthopper eggs |
| CN110724708B (en) * | 2019-10-16 | 2021-08-03 | 浙江大学 | Application of CvBV7-1 gene in reducing humoral immune response of drosophila melanogaster |
| CN111233993B (en) * | 2020-03-13 | 2020-12-01 | 广西壮族自治区水产科学研究院 | Prawn coupling antibacterial peptide and gene, acquisition method of prawn coupling antibacterial peptide, expression vector, recombinant bacterium and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810971A (en) * | 2005-09-13 | 2006-08-02 | 山东大学 | Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application |
| CN101333254A (en) * | 2007-06-29 | 2008-12-31 | 上海市上海中学 | Method for preparing drosophila melanogaster antimicrobial peptide |
| CN102766635A (en) * | 2012-05-25 | 2012-11-07 | 上海大学 | Construction method of human dcf1 gene transgenic drosophila melanogaster model |
-
2014
- 2014-03-04 CN CN201410075171.0A patent/CN103865933B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810971A (en) * | 2005-09-13 | 2006-08-02 | 山东大学 | Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application |
| CN101333254A (en) * | 2007-06-29 | 2008-12-31 | 上海市上海中学 | Method for preparing drosophila melanogaster antimicrobial peptide |
| CN102766635A (en) * | 2012-05-25 | 2012-11-07 | 上海大学 | Construction method of human dcf1 gene transgenic drosophila melanogaster model |
Non-Patent Citations (3)
| Title |
|---|
| Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens;R. Carballar-Lejarazu et al.;《Cell. Mol. Life Sci.》;20081031;第65卷(第19期);第3081-3092页，尤其是摘要、3085页左栏第2段至右栏第2段、第3088页左栏第1段-右栏第1段、图7B、表2 * |
| SNF1A转基因果蝇的构建;徐天宏 等;《复旦学报》;20030228;第42卷(第1期);第98-102页 * |
| 抗菌肽及其转基因动物的研究进展;郭向华;《临床和实验医学杂志》;20040331;第3卷(第1期);第47-50页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103865933A (en) | 2014-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103865933B (en) | The application of a kind of WAP gene in Drosophila transgenic | |
| Chandrakala et al. | Vibriosis in shrimp aquaculture a review | |
| Su et al. | Effects of antimicrobial peptides on serum biochemical parameters, antioxidant activity and non-specific immune responses in Epinephelus coioides | |
| CN100366726C (en) | Culture solution and preservation solution for coccidian oocyst | |
| CN100577806C (en) | Recombinant plasmid and engineering bacterium containing grass carp interferon gene and their application | |
| Mazon Suastegui et al. | Aquacultural homoeopathy: a focus on marine species | |
| De Schryver et al. | Convergent dynamics of the juvenile European sea bass gut microbiota induced by poly‐β‐hydroxybutyrate | |
| US11939576B2 (en) | Transgenic microalgae and use thereof as a feed for delivery of interfering RNA molecules | |
| Zhang et al. | Impact of Vibrio parahaemolyticus and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp Litopenaeus vannamei | |
| CN104322931B (en) | A kind of special feed additive for breeding laying duck | |
| Hettiarachchi et al. | Isolation of the bacterium, Vibrio harveyi from cultured shrimp, Penaeus monodon and production of vaccines against the bacterium | |
| Santhyia et al. | Molecular variations in Vibrio alginolyticus and V. harveyi in shrimp-farming systems upon stress | |
| CN106577381B (en) | Breeding method of excellent streptococcus agalactiae disease resistant strain of genetically improved farmed tilapia | |
| CN102295694A (en) | Antimicrobial polypeptide, and preparation method and application thereof | |
| CN101376675B (en) | Antimicrobial peptide | |
| CN103232950A (en) | Shewanellajaponica HS7 strain and use thereof | |
| KR20170001379A (en) | Inactivated vaccine composition against atypical Aeromonas salmonicida in rockfish | |
| CN104996337A (en) | Aquatic product culture disease prevention method | |
| CN103074362A (en) | Production process and application of antibacterial peptide used in animal feed | |
| CN102091089A (en) | Application of active burdock oligosaccharide to aquaculture | |
| CN110713955A (en) | Lactic acid bacteria and application thereof in aquaculture | |
| CN101007049A (en) | Application of nitric oxide donor and Chinese herbal preparation on nonspecific immune function of prawns | |
| CN115119781B (en) | Biological prevention and treatment method for ciliate disease | |
| WO2017079936A1 (en) | System expressing heterologous gene and use thereof | |
| CN109997794B (en) | Application of sulfadiazine in reproduction behavior of Wolbachia infected short-tube trichogramma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190103 Address after: 272000 The second one or two-storey business building in the north of the business building of section D along the river of Taibai Mall in Rencheng District, Jining City, Shandong Province Patentee after: Shandong Chentai Pharmaceutical Co., Ltd. Address before: 250022 No. 106 Jiwei Road, Shizhong District, Jinan City, Shandong Province Patentee before: University of Jinan |
|
| TR01 | Transfer of patent right |