CN102229664A - Recombinant melittin and application thereof - Google Patents

Abstract

The invention discloses a recombinant melittin and an application thereof, and belongs to the technical field of genetic engineering. An amino acid sequence of the recombinant melittin protein is shown in the formula SEQ ID NO.1. The recombinant melittin protein sequence can be utilized for a preparation of gene medicines utilized for preventing and treating fallopian tubal diseases of breeding hens, in other words, the recombinant melittin protein sequence is connected with a carrier pVAX1 to form a recombinant melittin expression carrier. In addition, a CMV promoter of the carrier pVAX1 is replaced by a chicken ovalbumin gene end 5' regulatory sequence which is an ov sequence, thus the recombinant melittin can be expressed specifically in chicken fallopian tubal tissue. The recombinant melittin and the gene medicines have the advantages of good effects of treating fallopian tubal diseases of breeding hens, safety, no toxicity and broad application prospect.

Description

A kind of reorganization mellitin and application thereof

Technical field

The present invention relates to gene engineering technology field, be specifically related to a kind of recombinate mellitin and the application in control kind of chicken salpingo disease thereof.

Background technology

The technology that DNA reorganization (Recombinant DNA) technology is promptly transformed and reconfigured DNA with artificial means.The adding that comprises the removal of fine cut to dna molecular, partial sequence, new sequence be connected, the dna molecular amplification, change cell over to duplicate breeding, screening, clone, evaluation and sequencing or the like, be the core of genetic engineering technique.Provide technical support for obtaining protein/polypeptide (following alleged " protein " and " polypeptide " are general).Existing many protein drugs, vaccine, diagnostic reagent adopt gene engineering method production.

The DNA medicine, it is that carrier for expression of eukaryon is advanced in the gene recombination with treatment meaning, directly transfers in animal or human's the cell again, gives expression to polypeptide or protein with therapeutic action, thereby reaches purposes such as preventing disease, treatment disease.It is different with traditional method for extracting, synthetic protein engineering medicine, need be at external synthetic and marking protein, need be at in-vitro separation and purifying yet, but gene plasmid is implanted in vivo, allow cell oneself goes to synthesize, separation and protein purification, and discharge into blood circulation voluntarily, in the part or distal site bring into play its function, and then reach purposes such as disease preventing and treating.The DNA drug targeting expressed carry out targeted therapy then can consider to enable particular expression in expression system promotor in the specific tissue.

It is cause of disease by catching an illness or the hen that carries disease germs is passed to the caused disease of filial generation that egg passes disease.Pass spreading of property disease owing to plant egg, the result causes commodity contaminated in hatching process for chick, and the chick that goes out shell is often dead, remaining also becomes the recessiveness chicken that carries disease germs.Owing to plant the generation that the chicken breeding enterprise generally adopts the method control disease of effective antibacterials of regularly throwing something and feeding, but Drug abuse causes Resistant strain to increase sharply.People render to the antimicrobial polypeptide aspect to sight.The expression amount of antimicrobial polypeptide in physical environment seldom, the chemical synthesis process cost is too high, generally be to obtain to change the problem that the antimicrobial polypeptide genetic animal had both solved the vivoexpression horizontal constraints by engineered means, exempted vivoexpression again after, the difficult problem of subsequent purification.

Taken to the engineered research of animal and plant at present, commentaries on classics antimicrobial polypeptide genetic animal and plant have appearred, report successfully imports the antimicrobial polypeptide gene in mouse, mosquito sialisterium and enteron aisle, tobacco, potato, paddy rice and the wheat (Huang Ying etc., 2007).Lazarev etc. (2002,2004,2005) studies show that, by directly express antimicrobial polypeptide (mellitin) in isolated cells or animal body, can effectively suppress the infection of germ (mycoplasma and chlamydozoan), reduce the animal dead rate.

In the transgenic research, the use of tissue-specific promoter can make the purpose foreign gene efficiently express in particular organization.The best expression organ of mosquito is sialisterium, enteron aisle, because these places are habitats of each etap of parasite, thus the propagation of inhibition malaria.By discovering to commentaries on classics antimicrobial polypeptide dna rat, immune system synthetic antimicrobial polypeptide can strengthen the resistibility of animal self to disease, these antimicrobial polypeptides have lethal effect (Bulet P et al, 2004) to bacterium, fungi, protozoon and abnormal cell.

At present, the research of domestic scholars mainly is as bio-reactor with chicken salpingo, expression alien gene, as Gao Bo etc. ovalbumin gene is cloned in the pGEM-T carrier, insert cosmid vector and be built into chicken salpingo specific expression carrier pOV, add reporter gene again and obtain recombinant vectors pOVlacZ, find behind the injection laying hen LacZGene is only expressed in the portion of expanding of injection chicken salpingo.Clone's chicken ov gene control region can effectively drive reporter gene at oviducal specifically expressing.(structure of chicken salpingo specific expression carrier and expression in vivo thereof, Chinese biological engineering impurity, the 23rd the 8th phase of volume of August in 2003,83-86).

Mellitin is the micromolecule polypeptide that a class derives from insect, is made up of 26 amino-acid residues, and molecular weight 2840 has biologic activity widely, can suppress the growth and breeding of more than 20 kind of Gram-positive and negative bacteria.But the hemolytic action of mellitin is strong, has limited its clinical application.There is the scholar that the molecular structure of mellitin is transformed, the 5th Val become Arg, the 15th Ala becomes Arg, deleted the 16th Leu(Zhao Ya China, understand south unexpectedly, the expression in pichia spp of the transformation of melittin molecule and gene thereof, Chinese biological engineering impurity, 2005,25(2): 45-48).Can keep anti-microbial activity through improved mellitin, hemolytic activity reduces simultaneously.Also there are some researches show, reorganization mellitin adenovirus carrier with alpha-fetoprotein (AFP) promotor structure, the propagation that can suppress AFP masculine liver cancer cell, and AFP negative HCC cell and normal liver cell are not obviously influenced (Li Bai etc., the effect of melittin gene recombinant adenovirus liver cancer apoptosis reducing, China's hepatopathy impurity, 2004,12(8): 453-455).More than research all only at human diseases, yet there are no report about being used for the treatment of kind of the genomic medicine of chicken salpingo disease.

Summary of the invention

The objective of the invention is to the chemosynthesis antibacterial peptide cost height according to kind of the chicken salpingo disease of treatment in the prior art, the defectives such as medicine shortage of biological method preparation, a kind of reorganization mellitin albumen for the treatment of kind of chicken salpingo disease is provided.

Another object of the present invention provides the genomic medicine by above-mentioned mellitin preparation.

Another purpose of the present invention provides the preparation method of said gene medicine.

The present invention realizes above-mentioned purpose by following technique means:

A kind of reorganization mellitin albumen (called after MLT) is characterized in that aminoacid sequence is shown in SEQ ID NO:1.The present invention transforms mellitin albumen, changes its Lys-7 into Arg-7, and Val-8 changes Gly-8 into and deleted the Leu-13 site, increases the purpose that the mellitin bacteriostatic activity reduces its hemolytic activity simultaneously to reach.At first, change Lys-7 into Arg, with the reinforcement positive charge, and Arg is the insensitive person of spiral; Secondly, all change Val-8 into Gly, break spiral, strengthen hydrophobic moment; Deletion Leu-13: from thermodynamic (al) angle analysis, peptide chain is long more, and folding required time is long more, also is unfavorable for and the combining of film, and the folding of Leu-13 and spiral has nothing to do, and deletes 13 and can significantly reduce haemolysis.

The proteic encoding gene of above-mentioned reorganization mellitin, nucleotide sequence is shown in SEQ ID NO:2.

The application of above-mentioned reorganization mellitin albumen in the medicine of preparation treatment kind of chicken salpingo disease.

Described kind of chicken salpingo disease is coli-infection disease, white dysentery Salmonella infection disease or infection of staphylococcus aureus disease.

A kind of reorganization mellitin expression vector is to be connected and composed by proteic encoding gene of above-mentioned reorganization mellitin and carrier pVAX1.Carrier pVAX1 purchases the company in invitrogen.A strong promoter pcmv is arranged, and the downstream has multiple clone site and length features of smaller to help the carrier transformation.Reorganization mellitin expression vector pVAX1-MLT keeps its strong promoter pcmv and inserts antibacterial peptide gene in the multiple clone site district on the basis of pVAX1 plasmid, make antibacterial peptide gene start under the regulation and control of pcmv by force.

On the basis of above-mentioned expression vector, we have done further improvement, promptly by chicken salpingo specific expression promoter chicken egg white (Ovalbumin, ov) 5 ' ending regulating sequence 1.1kb(GenBank accession number is J00895), be called for short the CMV promotor on the ov sequence replacement vector pVAX1, be connected structure with the proteic encoding gene of above-mentioned reorganization mellitin and form.Said gene medicine promptly recombinate multiplicative model figure such as Fig. 1 of mellitin expression vector.After doing such improvement, the antimicrobial polypeptide gene is placed under the control of chicken salpingo specifically expressing controlling element, realize the specifically expressing of mellitin at histoorgan, expression product is 5.36kD.

Utilize the uterine tube specific expression promoter to drive of the expression of antimicrobial polypeptide gene, make external source antimicrobial polypeptide gene in uterine tube, to be secreted in the egg white with other albumen at the uterine tubal epithelium cell.The antimicrobial polypeptide gene is expressed in the egg forming process, and be assembled into egg together.For suppressing egg biography property disease, improving the natality of chick and reducing the courses of infection rate provides a kind of genomic medicine.

Improved reorganization mellitin preparation of expression vectors method is characterized in that may further comprise the steps:

(1) amplification reorganization Melittin gene;

(2) reorganization Melittin gene in the step (1) is connected on the carrier pVAX1

(3) amplification chicken ovalbumin gene;

(4) step (3) gained chicken ovalbumin gene is connected on the carrier pMD18-T; Earlier chicken ovalbumin gene is checked order, just be used for next step test after order-checking is correct;

(5) step (2) products therefrom and step (4) products therefrom are recombinated after enzyme is cut, make up the pVAX1 recombinant vectors that contains reorganization Melittin gene, chicken ovalbumin gene, be the genomic medicine of treatment kind of chicken salpingo disease.

The method of the described amplification reorganization of step (1) Melittin gene is: add translation initiation sequence CGCCACC and initiator codon ATG at the proteic encoding gene 5 ' end of the described reorganization mellitin of claim 2,3 ' end adds 6 His, and adds restriction enzyme site at 5 ' end Nhe

, Bgl

, 3 ' end adds EcoR I and protection base ATT, as template, PCR is carried out in design primer sequence such as SEQ ID NO:3 ~ 4, is template with the PCR product, and design primer sequence such as SEQ ID NO:5 ~ 6 are carried out second and are taken turns PCR, and the PCR product is the reorganization Melittin gene.

The method of the described amplification chicken ovalbumin gene of step (2) is: with the total DNA of chicken salpingo is template, is primer with SEQ ID NO:7 ~ 8, carries out PCR, and the PCR product is chicken ovalbumin gene.

A kind of genomic medicine of preventing and treating the chicken salpingo disease comprises above-mentioned any one reorganization mellitin expression vector.

Compared with prior art, the present invention has following beneficial effect:

Reorganization mellitin albumen of the present invention has stronger bacteriostatic action, and the recombinant expression vector genomic medicine of structure can be brought into play its drug effect at chicken salpingo epithelium specifically expressing, and medicine itself do not have toxicity, and drug safety can be protected.

Description of drawings

Fig. 1. recombinant plasmid pOV1.1-MLT physical model figure;

Fig. 2. the PCR electrophoresis detection figure of MLT gene, wherein M is Marker, 1,2 is pcr amplification product;

Fig. 3. the double digestion electrophorogram of pVAX1-MLT.Wherein M is Marker, and 1,2 cut the result for pVAX1-MLT recombinant plasmid enzyme;

Fig. 4. the physical model figure of expression plasmid pVAX1;

Fig. 5. reorganization mellitin expression vector pVAX1-MLT physical model figure;

Fig. 6. chicken egg white upstream regulatory sequence ov PCR electrophoresis detection figure, wherein M is Marker, 1,2 is the PCR product of ovalbumin upstream regulatory sequence;

Fig. 7. pMD18-T-OV bacterium colony PCR identifies electrophorogram, and M is Marker, and 1 ~ 11 is the bacterium colony PCR product of pMD18-T-OV transformant;

Fig. 8. pOV1.1-MLT bacterium colony PCR identifies electrophorogram, and M is Marker, and 1 ~ 4 is pOV1.1-MLT transformant bacterium colony PCR product;

Fig. 9. recon pOV1.1-MLT double digestion is identified electrophorogram, and M is Marker, and 1 cuts product for the enzyme of recon pOV1.1-MLT;

Figure 10. three kinds of plasmids are to the antibacterial result of pathogenic bacteria, and wherein a is the transfection empty plasmid, and b is transfection pVAX1-MLT, and c is transfection pOV _1.1-MLT, horizontally-arranged 1 is to intestinal bacteria K ₁₂D ₃₁, 2 is that white dysentery Salmonellas, 3 is streptococcus aureus;

Figure 11. Tricine-SDS PAGE detects CHO/Vero cell expression product figure as a result, and M is Marker, and 1 is transfection empty plasmid sample, and 2 are transfection pOV1.1-MLT sample, and 3 are transfection pVAX1-MLT sample;

Figure 12. detect expressing cho cell product figure, 1 is pVAX1-MLT plasmid PCR product, and 2 ~ 3 is empty plasmid transfectional cell RT-PCR product, and 4 ~ 5 is pVAX1-MLT plasmid transfection cell RT-PCR product, and 6 ~ 7 is pOV _1.1-MLT transfectional cell RT-PCR product;

Figure 13. detect Vero cell expression product figure, 1 is pVAX1-MLT plasmid PCR product, and 2 ~ 3 is pOV _1.1-MLT transfectional cell RT-PCR product, 5 ~ 6 is pVAX1-MLT transfectional cell RT-PCR product;

Figure 14. Chinese hamster ovary celI immunofluorescence result (200 *), a are pVAX1-MLT, and b is pOV _1.1-MLT;

Figure 15. Vero cellular immunofluorescence result (200 *), a are pVAX1-MLT, and b is pOV _1.1-MLT;

Figure 16. chicken histoorgan frozen section immunofluorescence is observed, and a is the negative control of injection empty plasmid;

Figure 17. chicken histoorgan frozen section immunofluorescence is observed, and b is injection recombinant plasmid pVAX1-MLT;

Figure 18. chicken histoorgan frozen section immunofluorescence is observed, and c is injection recombinant plasmid pOV _1.1-MLT;

Figure 19. (400 *) are observed in dyeing to the HE of chicken tissue freezing section, the left side: the tissue that negative control pVAX1 handles, the right: the tissue that recombinant plasmid pVAX1-MLT handles, (order is from top to bottom: heart, liver, spleen, kidney, muscle, ovary and uterine tube).

Embodiment

Embodiment 1. preparation reorganization mellitin and encoding genes thereof

1. transform the mellitin aminoacid sequence

On existing research basis, former mellitin aminoacid sequence is transformed to reach the purpose that increase mellitin bacteriostatic activity reduces its hemolytic activity simultaneously.

Original mellitin aminoacid sequence is shown in SEQ ID NO:9;

Improved aminoacid sequence shown in SEQ ID NO:1, called after MLT.

Be about to Lys-7 and change Arg-7(reinforcement positive charge into, and Arg is the insensitive person of spiral), Val-8 changes Gly-8(into and breaks spiral, strengthen hydrophobic moment) and deleted the Leu-13 site (from thermodynamic (al) angle analysis, peptide chain is long more, and folding required time is long more, also is unfavorable for and the combining of film, the folding of Leu-13 and spiral has nothing to do, and deletes 13 and can significantly reduce haemolysis).

2. obtain the encoding gene of reorganization MLT

Infer the encoding gene of mellitin according to improved aminoacid sequence and chicken preference codon, its nucleotide sequence such as SEQ ID NO:2, add Kozak translation initiation sequence (CGCCACC) and initiator codon ATG at its 5 ' end, 3 ' end adds that 6 Histidines are used for detecting, and adds restriction enzyme site 5 ' end at two ends Nhe

, Bgl

, 3 ' end EcoR I and protection base ATT; design 4 primer sequence SEQ ID NO:3 ~ 6; carry out PCR by following program: with above-mentioned mellitin coding gene sequence through modification is template; each 1 μ L of primer SEQ ID NO:3 ~ 4 (20 μ mol/ L); dNTPs (10mmol/L) 1 μ L, 10 * buffer (Mg ²⁺) 5 μ L, rTaq DNA polysaccharase (2.5U/ μ L) 1 μ L adds ddH ₂O to 50 μ L.As template, carry out the pcr amplification second time for primer with the PCR product that draws with SEQ ID NO:5 ~ 6.PCR reaction system composed as follows: 10 * buffer, 5 μ L, dNTPs 2 μ L, each 2 μ l of primer (20 μ mol/L), rTaq DNA polysaccharase (2.5U/ μ L) 0.5 μ L, Template 1 μ L, ddH ₂O mends to 50 μ L.PCR response procedures: 94 ℃ of pre-sex change 2min, 1 circulation; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 40s, 30 circulations; Extend 5min after 72 ℃.

Amplification such as Fig. 2, PCR product can be observed the specific band at 100bp ~ 250bp when 1.5% agarose gel electrophoresis detects, be the MLT gene.

Embodiment 2. makes up reorganization mellitin expression vector pVAX1-MLT

The MLT gene of embodiment 1 gained is used NheI and EcoR I carries out double digestion digestion, 37 ℃ of effect 3h.Reaction system is as follows: 10 * buffer M, 5 μ L, NheI 1 μ L, EcoR I 1 μ L, MLT gene 25 μ L, ddH ₂O mends to 50 μ L, simultaneously the pVAX1 plasmid is used restriction endonuclease equally NheI and EcoRI carries out enzyme and cuts digestion, 37 ℃ of effect 3h.Reaction system is as follows: 10 * buffer M, 5 μ L, NheI 1 μ L, EcoR I 1 μ L, pVAX1 plasmid 20 μ L, ddH ₂O mends to 50 μ L, uses the CIAP dephosphorylation simultaneously.Reaction solution after enzyme is cut is all used 1.5% agarose gel electrophoresis, downcuts the segmental band of purpose under ultraviolet lamp, and blended rubber reclaims the target DNA fragment after test kit recovery enzyme is cut.Under the effect of T4 ligase enzyme, the pVAX1 carrier of digestion is connected for 4 ℃ with the MLT gene that has digested and spends the night.Reaction system is as follows: 10 * connecting buffer 1 μ L, the MLT gene enzyme cuts back to close product 7 μ L, and the pVAX1 plasmid reclaims product 1 μ L, T4 dna ligase 1 μ L.Reaction finishes the back and obtains to connect product recombinant expression vector pVAX1-MLT.

To connect product and transform DH5 α competent cell, the picking positive colony continues positive colony to cultivate at 37 ℃ with the kana substratum about 3ml, extracts plasmid then.Use restriction enzyme NheI and EcoR I carries out enzyme and cuts evaluation, electrophoresis result such as Fig. 3.Send and be accredited as the male recombinant plasmid and carry out sequencing to AudioCodes biotech company.The correct recombinant plasmid that checks order is the plasmid pVAX1-MLT of new structure.The model diagram of plasmid pVAX1 and recombinant plasmid pVAX1-MLT is seen Fig. 4,5.

Embodiment 3. makes up reorganization mellitin expression vector pOV1.1-MLT

1. the amplification of chicken egg white ov sequence

According to the chicken ovalbumin gene of announcing on the ncbi database (Chicken Ovalbumine, ov) full nucleotide sequence length is 9206bp, code length is the ripe mRNA of 1872bp.(the GenBank accession number: J00895) designing the upstream and downstream primer, and added restriction site at the fragment two ends, as SEQ ID NO:7 and SEQ ID NO:8, is template with the total DNA of chicken salpingo, with above primer amplified ov sequence.PCR reaction system composed as follows: 10 * buffer, 2.5 μ L, dNTPs 2 μ L, Primers(20 μ mol/L) each 1 μ L, rTaq DNA Polymerase (2.5U/ μ L) 0.5 μ L, Template 0.5 μ L, ddH ₂O mends to 25 μ L.PCR response procedures: 94 ℃ of pre-change 5min, 1 circulation; 94 ℃ of 40s, 55 ℃ of 40s, 72 ℃ of 80s, 30 circulations; Extend 10min after 72 ℃.With above-mentioned product is template, carries out PCR again one time, and every add-on doubles in the system, and cumulative volume is 50 μ L.

Amplification such as Fig. 6.The PCR product can be observed the specific band about 1000bp when 1.5% agarose gel electrophoresis detects.The ov sequence length is 1.1kb, and the PCR product can be observed a little higher than 1000bp band of specific band size when agarose gel electrophoresis detects, be the ov sequence.

2. the ov cloned sequence connects the T carrier

Ov sequence PCR product and pMD18-T Vector(TAKARA company with purifying) be connected.Reaction system is: Solution I 5 μ L, the ov sequence PCR product 4 μ L of purifying, pMD18-T Vector 1 μ L.After mixing is centrifugal, spend the night in 4 ℃ of connections.Connect product and be directly used in conversion DH5 α competent cell, the picking positive colony continues positive colony to cultivate at 37 ℃ with the LA substratum about 3ml, extracts plasmid then.The method of cutting with enzyme is carried out enzyme and is cut evaluation, qualification result such as Fig. 7.Send and be accredited as the male recombinant plasmid and carry out sequencing to AudioCodes biotech company.The correct recombinant plasmid that checks order is pMD18-T-OV.

3. make up reorganization mellitin expression vector POV1.1-MLT

For the antimicrobial polypeptide gene is placed under the control of chicken salpingo specifically expressing controlling element, the promotor of pVAX1-MLT plasmid is replaced with the ov regulating and controlling sequence.Recombinant plasmid pMD18-T-OV and pVAX1-MLT are used respectively MluI and NheI carries out double digestion, 37 ℃ of effect 3h.Reaction system is as follows: 10 * buffer M, 5 μ L, Nhe I 1 μ L, MluI, recombinant plasmid 20 μ L, ddH ₂O mends to 50 μ L, uses the CIAP dephosphorylation simultaneously.Reaction solution after enzyme is cut is all used 1.5% agarose electrophoresis, downcuts the segmental band of purpose under ultraviolet lamp, and blended rubber reclaims the target DNA fragment after test kit recovery enzyme is cut.Under the effect of T4 ligase enzyme, the ov sequence fragment that digests purifying is connected for 4 ℃ with the pVAX1-MLT vector plasmid that has digested spends the night.Reaction system is as follows: 10 * connection buffer, 1 μ L, ov sequence fragment 7 μ L, pVAX1-MLT 1 μ L, T4 dna ligase 1 μ L.

To connect product and transform DH5 α competent cell, and get the coating of bacterium liquid at last and contain on the LB nutrient agar of 100 μ g/mL kana, cultivate 12 ~ 16h for 37 ℃.Picking list colony inoculation 3 mL contain in the LB nutrient solution of 50 μ g/mL kana on the flat board, after 12h is cultivated in 37 ℃ of joltings, get bacterium liquid and carry out the PCR detection with primer SEQ ID NO:7 ~ 8, set up blank and positive control simultaneously, reaction system is as follows: 10 * PCR buffer, 1.5 μ L, bacterium liquid 1 μ L, dNTPs 0.5 μ L(2.5mM), each 0.5 μ L of Primers (20 μ mol/ L), rTaq DNA Polymerase 0.25 μ L (2.5U/uL), ddH ₂O mends to 15 μ L.The ov sequence that increases according to a conventional method, reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 40s, 55 ℃ of annealing 40s, 72 ℃ are extended 80s, circulate 30 times, and last 72 ℃ are extended 10min.The PCR product with 1.5% sepharose (contain 0.5 μ g/mL ethidium bromide, EB) electrophoresis detection PCR product carries out electrophoresis under the voltage of 100V, behind the 20min under ultraviolet lamp observations, as Fig. 8.The picking positive colony, positive colony is cultivated at 37 ℃ of 220rpm overnight shakings with the Kana substratum about 3ml, inferior daily E.Z.N.A.Plasmid Minipreps Kit extracts plasmid, operate the extraction plasmid by the test kit specification sheets, the method of cutting with enzyme is carried out enzyme and is cut evaluation, electrophoresis result such as Fig. 9.Send to be accredited as the male recombinant plasmid and to carry out sequencing, obtain the chicken salpingo specific expression carrier to AudioCodes biotech company.The correct recombinant plasmid that checks order is the antimicrobial polypeptide gene chicken salpingo specific expression carrier of new structure, called after pOV1.1-MLT.

Embodiment 4 recombinant vectors transfection Vero and Chinese hamster ovary celIs

The bacterium liquid of the recombinant plasmid pVAX1-MLT, the pOV1.1-MLT that obtain in embodiment 2 and 3 and pVAX1 carrier is streak culture on the kana culture plate, in 37 ℃ of incubators, spend the night.Next day, picking list bacterium colony was cultivated at 37 ℃ of 220rpm overnight shakings with the kana substratum of 3ml.Afterwards with the triangular flask that 400ml kana substratum the is housed enlarged culturing of in 37 ℃ of incubators, spending the night.The big upgrading grain test kit large quantity extracting plasmid of the inferior daily day biological company limited of root.Detect the concentration of plasmid, put-20 ℃ of preservations.When treating that Vero, Chinese hamster ovary celI reach the optimum growh state, inoculate 0.5 ~ 2 * 10 respectively ⁵The cell of/hole number in 6 orifice plates, when treating that cell reaches 90 ~ 95% degree of converging, with recombinant plasmid pVAX1-ML and pOV1.1-MLT with liposome-mediated transfection Vero cell and Chinese hamster ovary celI respectively.Distinguish two kinds of cells of transfection in contrast together with empty plasmid pVAX1.Hatch 6h for 37 ℃, be replaced by perfect medium and continue to cultivate, add estradiol 10 simultaneously ^-7Mol/L, Kendall compound 10 ^-6Mol/L and Regular Insulin 40 μ g/L.After cultivating 24h, 48h, place observation inverted microscope under, and the collecting cell supernatant ,-80 ℃ of preservations are used to detect the bacteriostatic action of cell culture supernatant, detect cell expression product with Tricine-SDS PAGECHO.Detect the recombinant vectors cells transfected with RT-PCR and cellular immunofluorescence afterwards.

1. the detection of the bacteriostatic action of cell culture supernatant

With 0.22 μ m membrane filtration, each sample is got 4mL, is lyophilized into powder, and is heavy molten, standby with the not freeze dried nutrient solution 100 μ L of residue with the cell culture supernatant collected.With Gram-negative bacteria e. coli k12 D31( Escherichia coli), the white dysentery Salmonellas ( Salmonella pullorum) and the gram-positive microorganism streptococcus aureus ( Staphylococcus aureus) be the cause of disease indicator, adopt Microdilution methods analyst expression product external activity: the bacterium liquid (1 * 10 of 100 μ L ⁵CFU/well)+100 μ L protein solution.After 37 ℃ of above-mentioned mixed solutions are cultivated 2h, get 1 μ L and join in the semi-solid LB substratum that has melted (50 ~ 60 ℃), pour in the thin LB flat board of existing one deck 37 ℃ of overnight incubation behind the mixing into.Residual mixed liquor continues to cultivate 12h, measures OD600nm.Dull and stereotyped cultivation results such as Figure 10, in e. coli k12 D31, white dysentery Salmonellas and staphylococcus aureus, the reorganization mellitin is the strongest to the inhibition of streptococcus aureus.

2. Tricine-SDS PAGE detects the CHO/Vero cell expression product.

Cell culture fluid is removed in suction, remain 500 μ L, the cell of transfection 48h scraped gently with cell from 6 orifice plates scrape, 4 ℃ of centrifugal 15min of 4000r/min draw supernatant to remaining 50 μ L, re-suspended cell, add 5 * SDS loading buffer more respectively, boil 5min, the centrifugal 15min of 12000rpn, get supernatant liquor, obtain the total protein sample.Each layer of preparation 16.5%Tricine-SDS-PAGE gel adds cathode buffer liquid (0.2mol/L Tris-HCl pH8.9) and cathode buffer liquid (100mmol/L Tris-HCl respectively, 100mmol/L Tricine, 0.1%SDS), get above-mentioned sample point sample 15 μ L.Voltage stabilizing 40mV electrophoresis.After treating that albumen enters at interval glue, can transfer to 100mV, stop electrophoresis from 1cm place, glue bottom to indicator.The careful gel that takes out is placed in the stationary liquid fixedly 45min earlier, then with the staining fluid dyeing 30min that contains coomassie brilliant blue R250.Change the destainer decolouring rinsing of slightly vibrating, till the gel background transparent, take a picture and analyze, experimental result such as Figure 11 have a lighter swimming band near Marker 5kD, conform to the intended purposes molecular weight of albumen (MLT:5.36kD).The sample strip of pVAX1-MLT transfection is comparatively obvious, pOV1.1-MLT transfection lighter.

3. RT-PCR detects the recombinant vectors cells transfected

Press the working instructions that Trizol Reagent RNA extracts test kit, extract the total RNA in the transfectional cell.After the cell total rna that extracts was hatched 30min with 37 ℃ of RNase-Free DNase ,-80 ℃ of preservations were standby after being directly used in reverse transcription-polymerase chain reaction (RT-PCR) or adding RNase Inhibitor by a certain percentage.Press RQ1 RNase-Free DNase explanation and handle RNA, RT-PCR method amplifying target genes is adopted in reaction, reaction system is: 2 * One Step RNA PCR buffer, 25 μ L, each 1 μ L of primer SEQ ID NO:5 and SEQ ID NO:6, RNA 5 μ L, 1 step Enzymes Mix, 2 μ L, RNase Free dH ₂O mends to 50 μ L.With above-mentioned reaction system mixing, centrifugal, on PCR Express Gradient PCR instrument, carry out following response procedures: 50 ℃ of 30min, 94 ℃ of pre-sex change 2min, a circulation; 94 ℃ of 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations.Amplified production detects with 1.5% agarose gel electrophoresis, for Chinese hamster ovary celI RT-PCR product experimental result such as Figure 12; For Vero cell RT-PCR product experimental result such as Figure 13.Compare and can find out: the promotor of pVAX1 all has to start in CHO and Vero cell to be expressed, but the promotor that has replaced to the chicken salpingo specifically expressing only has startup to express in CHO.

4. cellular immunofluorescence detects the recombinant vectors cells transfected.

The preparation cell climbing sheet behind transfection 48h, takes out cell climbing sheet earlier, after the slight rinsing of PBS, places 37 ℃ of PBS 10 ~ 30min, and the residual nutrient solution of dialysing is washed 3 times with PBS again, each 5min.With the fixing 30min of 4% Paraformaldehyde 96, wash 3 times each 10min again with PBS.With saturatingization of histocyte punching liquid 2 ~ 5min, PBS washes 3 times, each 10min.Add confining liquid (5%BSA), room temperature sealing 60min.Exhaust confining liquid, add the good anti-Histidine Tag(1:50 of dilution immediately), 4 ℃ of overnight incubation.Reclaim one and resist, PBS washes 3 times, each 5min.Fluorescent mark two anti-(1:100), the wet box lucifuge of room temperature is hatched 1h; PBS washes 3 times, each 5min.Envelope is mounted the agent mounting, and microscopy is taken pictures.Chinese hamster ovary celI immunofluorescence detected result and Vero cellular immunofluorescence detected result such as Figure 14 and Figure 15, coloration result shows that pVAX1-MLT, pOV1.1-MLT all can express in Chinese hamster ovary celI, expression product mainly is at endochylema; In the Vero cell, have only the pVAX1-MLT sample can observe a spot of cell colour developing.

Embodiment 6 imports the chicken expression in vivo with the recombinant vectors of the ov promotor that driven is expressed

Recombinant plasmid pVAX1-MLT, pOV1.1-MLT and empty plasmid pVAX1 be through the mixed of liposome LipoFu2008(with 1:1, the static 20min of room temperature) after the parcel, go in the chicken body by the wing intravenous injection.3 female numb chickens (it is suitable to select body weight) that are in egg-laying peak 150 day age of every kind of plasmid injection, 500 μ g/ time/injected two days continuously.At totally 3 chest muscles injection 0.3mg oestrogenic hormon on injection plasmid the day before yesterday and same day.From the collection day before yesterday egg of injection for the first time, treat the 4th day after the injection for the second time, every sample selects a chicken to slaughter, and gets each internal organs (heart, liver,spleen,kidney, ovary, uterine tube and muscle).Each internal organs tissue of chicken with obtaining extracts total RNA respectively, and product carries out reverse transcription PCR, testing goal band MLT sequence, and adopt frozen section immunofluorescence technique rapid detection simultaneously and carry out the pathology detection, method is as follows:

Immunofluorescence detects: slaughter each internal organs of acquisition, drop into that liquid nitrogen is anxious to be frozen the several seconds, take out Zhi Yu – and spend the night for 80 ℃, carried out frozen section, fluorescent dye simultaneously in second day.Staining procedure is as follows: with frozen section acetone fixed 10min; PBS washes 2 times, each 5min; 10% normal goats serum sealing 30min; Add an anti-Histidine Tag(1:200) 4 ℃ of overnight incubation; PBS washes 3 times, each 5min; The wet box lucifuge of fluorescent mark two anti-(1:200) room temperatures is hatched 1h; PBS washes 3 times, each 5min; The wet box lucifuge of DAPI room temperature is hatched 5min; PBS washes once, 5min; 80% glycerine mounting, microscopy is taken pictures.

Pathology detect: after will having injected the sample frozen section of the sample of empty plasmid and pVAX1-MLT, carry out Hematorylin-Yihong (HE) dyeing, observe whether pathological change takes place, staining procedure is as follows: 1. frozen section haematoxylin dyeing 2min; 2. after washing raffinate, the differentiation several seconds; 3. wash, return indigo plant; 4. 1% Yihong dyeing 5min; 5. wash raffinate; 6. conventional ethanol dehydration, dimethylbenzene is transparent; 7. mounting, microscopy is observed.

Detected result: histogenic immunity fluoroscopic examination result such as Figure 16,17,18, can see the band that a rectangular apparent ruddiness is arranged intuitively from picture figure c-uterine tube, this place is the uterine tubal epithelium cell, shows that MLT is at the uterine tubal epithelium cell expressing.

Pathology detected result such as Figure 19 have injected the internal surface of recombinant plasmid and the organ indistinction of negative control (empty plasmid) as can be seen, prove its free of toxic effects.

SEQUENCE LISTING

＜110〉Agricultural University Of South China

＜120〉a kind of reorganization mellitin and application thereof

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<170> PatentIn version 3.3

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Met Gly Ile Gly Ala Val Leu Arg Gly Leu Thr Thr Gly Pro Ala Leu

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ggaattggag ctgtgctgcg cggactgaca acaggaccag ctctgatttc ttggattaaa 60

cgcaaacgcc aacag 75

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tcagagctgg tcctgttgtc agtccgcgca gcacagctcc aattc 45

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Claims (10)

1. a reorganization mellitin albumen is characterized in that aminoacid sequence is shown in SEQ ID NO:1.

2. the proteic encoding gene of the described reorganization mellitin of claim 1, nucleotide sequence is shown in SEQ ID NO:2.

3. the application of the described reorganization mellitin of claim 1 albumen in the medicine of preparation treatment kind of chicken salpingo disease.

4. application according to claim 3 is characterized in that described kind of chicken salpingo disease is coli-infection disease, white dysentery Salmonella infection disease or infection of staphylococcus aureus disease.

5. a reorganization mellitin expression vector is characterized in that it being to be the carrier that sets out with carrier pVAX1, contains the proteic encoding gene of the described reorganization mellitin of claim 2.

6. reorganization mellitin expression vector is characterized in that it being CMV promotor by on the chicken salpingo specific expression promoter ov sequence replacement vector pVAX1, and the downstream connects the proteic encoding gene of the described reorganization mellitin of claim 2 and makes up and form.

7. the described reorganization mellitin of claim 6 preparation of expression vectors method is characterized in that step is as follows:

(1) amplification reorganization Melittin gene;

(2) reorganization Melittin gene in the step (1) is connected on the carrier pVAX1;

(3) amplification chicken ovalbumin gene;

(4) step (3) gained chicken ovalbumin gene is connected on the carrier pMD18-T;

(5) step (2) products therefrom and step (4) products therefrom are recombinated after enzyme is cut, make up the pVAX1 recombinant vectors that contains reorganization Melittin gene, chicken ovalbumin gene, be the genomic medicine of treatment kind of chicken salpingo disease.

8. according to the preparation method of the described genomic medicine of claim 7, the method that it is characterized in that the described amplification reorganization of step (1) Melittin gene is: add translation initiation sequence CGCCACC and initiator codon ATG at the proteic encoding gene 5 ' end of the described reorganization mellitin of claim 2,3 ' end adds 6 His, and adds restriction enzyme site at 5 ' end Nhe

, Bgl

, 3 ' end adds EcoR I and protection base ATT, as template, PCR is carried out in design primer sequence such as SEQ ID NO:3 ~ 4, is template with the PCR product, and design primer sequence such as SEQ ID NO:5 ~ 6 are carried out second and are taken turns PCR, and second takes turns the PCR product is the reorganization Melittin gene.

9. preparation method according to claim 7, the method that it is characterized in that the described amplification chicken ovalbumin gene of step (3) is: with the total DNA of chicken salpingo is template, with SEQ ID NO:7 ~ 8 is primer, carries out PCR, and the PCR product is chicken ovalbumin gene.

10. a genomic medicine of preventing and treating the chicken salpingo disease is characterized in that comprising arbitrary reorganization mellitin expression vector in claim 5 or 6.

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