CN102816788A - Method for expressing eimeria acevulina 3-1E protein in lactococcus lactis - Google Patents
Method for expressing eimeria acevulina 3-1E protein in lactococcus lactis Download PDFInfo
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- CN102816788A CN102816788A CN2012103025853A CN201210302585A CN102816788A CN 102816788 A CN102816788 A CN 102816788A CN 2012103025853 A CN2012103025853 A CN 2012103025853A CN 201210302585 A CN201210302585 A CN 201210302585A CN 102816788 A CN102816788 A CN 102816788A
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Abstract
The invention relates to a method for expressing an eimeria acevulina 3-1E protein in lactococcus lactis, and relates to a method for expressing an eimeria acevulina 3-1E protein in order to solve the problem that the eimeria acevulina sporozoite/merozoite stage surface antigen 3-1E can not be expressed in the lactococcus lactis. The method comprises the following steps of optimizing an eimeria acevulina 3-1E gene sequence according to the preference of the condon of the lactococcus lactis, inserting a cloning vector and transforming escherichia coli cells to obtain positive transformants; carrying out enzyme digestion and connection of the positive transformants and a lactic acid bacteria expression vector, and constructing a recombinant expression vector; transforming competent lactic acid bacteria cells, and identifying; and culturing and inducing recombinant bacteria to complete the expression of the 3-1E protein in the lactococcus lactis NZ9000. The artificially synthesized codon-optimized 3-1E protein is successfully expressed in the lactococcus lactis. The method provided by the invention is used for the prevention field of coccidiosis.
Description
Technical field
The present invention relates to the proteic method of a kind of expression chicken heap type Eimeria 3-1E.
Background technology
The chicken Eimeria is one of important diseases of serious harm aviculture development.The chicken coccidia can cause in the reproductive process epithelial cell that damage and inflammatory reaction take place in vivo, and extensive injuries can cause that chicken suffers from diarrhoea, dehydration, and weight loss, proctoptosis, dysentery etc. are clinical disease seriously, can cause chicken death sometimes.The financial loss that the whole world causes because of coccidiosis every year is about 3,000,000,000 dollars, and wherein 80% is because the chicken weightening finish descends, feed conversion rate reduces and immediate causes such as death, and the 20%th, because the expense of chemicals research.Verified: frequently use chemicals can cause the appearance of resistance worm strain, drug residue can bring the animal derived food security problems.The high cost of novel against-coccidia medicament research and development makes pharmacy corporation lose interest in to developing new anticoccidial drug.Therefore the prevention of coccidiosis has become assistant officer's important topic to be solved with control.See that from research tendency the prevention of worm seedling will develop into the main means of control coccidiosis of chicken gradually.
Milk-acid bacteria is a common probiotic bacterium in the humans and animals enteron aisle, is acknowledged as security level (GRAS) mikrobe.Milk-acid bacteria is as the desirable strain safety of expressing foreign protein, nontoxic, and immunization route is simple.Milk-acid bacteria is different from other as expression vector and is not belonged to the living vaccine carrier (like attenuation salmonella, intestinal bacteria and poxvirus etc.) in the safety range.Lactic acid bacteria vector itself can not cause that strong immunization replys, and can utilize this carrier that animal is carried out repeatedly oral immunity.
The 3-1E gene is the surface antigen in chicken Eimeria sporozoite and merozoite stage; Molecular weight of albumen is 18-27ku; Sequence total length 1086bp; 3-1E albumen is made up of 170 amino acid, between Eimeria tenella (E.tenella), heap type Eimeria (E.acervulina) and murder by poisoning Eimeria (E.necatrix), very high homology is arranged all, can be used as the candidate gene of recombinant vaccine.But still there is not the report of in lactococcus lactis ssp, expressing chicken Eimeria sporozoite/merozoite stage surface antigen 3-1E at present both at home and abroad.
Summary of the invention
The present invention is the problem that will solve the chicken Eimeria sporozoite that is beyond expression in the present lactococcus lactis ssp/merozoite stage surface antigen 3-1E, and a kind of proteic method of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing is provided.
The present invention expresses the proteic method of chicken heap type Eimeria 3-1E in lactococcus lactis ssp, carry out according to the following steps:
One, chicken being piled type Eimeria 3-1E gene order is optimized according to the codon preference of lactococcus lactis ssp; Obtain the 3-1E gene of synthetic; And its 5 ' end with 3 ' end introduces the recognition sequence of BamH 1 and Xba 1 respectively; Be connected and transformed into escherichia coli DH5 α competent cell screening positive transformant pUC57-3-1E afterwards with cloning vector pUC57;
Two, pUC57-3-1E and intestinal bacteria-lactic acid bacteria shuttle expression vector pTX8048 behind BamH 2 and Xba2 double digestion, are reclaimed 3-1E fragment and carrier segments respectively respectively, after the T4 dna ligase connects, make up recombinant expression vector pTX8048-3-1E;
Three, pTX8048-3-1E is transformed in the competent cell of lactococcus lactis ssp NZ9000 through electricity; Obtain containing the positive recombination lactic acid lactococcus spp NZ9000/pTX8048-3-1E of recombinant plasmid through screening; From positive recombination lactic acid lactococcus spp, extract plasmid with plasmid extraction kit; To the 3-1E gene order design primer of synthetic in the step 1, carry out PCR respectively and identify afterwards, enzyme is cut and is identified and sequencing;
Four, NZ9000/pTX8048-3-1E is inoculated in the GM17 liquid nutrient medium that contains paraxin, after 30 ℃ of anaerobism overnight cultures, transfers with 1: 50 volume ratio and to carry out amplification cultivation in the GM17 liquid nutrient medium that contains paraxin, 30 ℃ of anaerobism are cultured to OD
600Be 0.4～0.6, the nisin with 10ng/ml continues inducing culture 3h afterwards, has promptly accomplished and in lactococcus lactis ssp NZ9000, has expressed 3-1E albumen.
Chicken Eimeria sporozoite is invaded in the body through the intestinal mucosa epithelial cell, and a series of secretion antigens on sporozoite surface can stimulate infected chicken to produce immunne response.The immunne response that milk-acid bacteria excited that can express foreign protein is similar with the route of entry of coccidia, can excite animal body to produce effective enteron aisle protective immune response.
The present invention expresses chicken heap type Eimeria 3-1E albumen in lactococcus lactis ssp, solved other a series of lactic acid bacteria expression vectors of application and all failed to express the proteic problem of 3-1E.This will establish solid basis for the milk-acid bacteria oral vaccine research of host bacterium to developing from now on based on chicken source enteron aisle symbiosis milk-acid bacteria.The present invention is with the expressing in lactococcus lactis ssp NZ9000 through the success of the 3-1E of synthetic albumen after codon optimized.
Description of drawings
Fig. 1 is PCR qualification result figure in the step 3 of embodiment; Fig. 2 cuts qualification result figure for enzyme in the step 3 of embodiment; Fig. 3 carries out Western blot analytical results figure for extracting bacterial protein among the embodiment.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: this embodiment is expressed the proteic method of chicken heap type Eimeria 3-1E in lactococcus lactis ssp, carry out according to the following steps:
One, chicken being piled type Eimeria 3-1E gene order is optimized according to the codon preference of lactococcus lactis ssp; Obtain the 3-1E gene of synthetic; And its 5 ' end with 3 ' end introduces the recognition sequence of BamH 2 and Xba 2 respectively; Be connected and transformed into escherichia coli DH5 α competent cell screening positive transformant pUC57-3-1E afterwards with cloning vector pUC57;
Two, pUC57-3-1E and intestinal bacteria-lactic acid bacteria shuttle expression vector pTX8048 behind BamH 3 and Xba3 double digestion, are reclaimed 3-1E fragment and carrier segments respectively respectively, after the T4 dna ligase connects, make up recombinant expression vector pTX8048-3-1E;
Three, pTX8048-3-1E is transformed in the competent cell of lactococcus lactis ssp NZ9000 through electricity; Obtain containing the positive recombination lactic acid lactococcus spp NZ9000/pTX8048-3-1E of recombinant plasmid through screening; From positive recombination lactic acid lactococcus spp, extract plasmid with plasmid extraction kit; To the 3-1E gene order design primer of synthetic in the step 1, carry out PCR respectively and identify afterwards, enzyme is cut and is identified and sequencing;
Four, NZ9000/pTX8048-3-1E is inoculated in the GM17 liquid nutrient medium that contains paraxin, after 30 ℃ of anaerobism overnight cultures, transfers with 1: 50 volume ratio and to carry out amplification cultivation in the GM17 liquid nutrient medium that contains paraxin, 30 ℃ of anaerobism are cultured to OD
600Be 0.4～0.6, the nisin with 10ng/ml continues inducing culture 3h afterwards, has promptly accomplished and in lactococcus lactis ssp NZ9000, has expressed 3-1E albumen.
Cloning vector pUC57 buys from Nanjing Genscript Biotechnology Co., Ltd. in the step 1.
Intestinal bacteria in the step 2-lactic acid bacteria shuttle expression vector pTX8048 is at " Microbial Cell Factories; 2011; 10:66doi:10.1186/1475-2859-10-66; Expanding the molecular toolbox for Lactococcus lactis:construction of an inducible thioredoxin gene fusion expression system " i.e. " microorganism cells factory; 2011; 10:66 doi:10.1186/1475-2859-10-66, the expansion of the lactococcus lactis ssp molecular tool box-derivable amalgamation and expression system that contains thioredoxin gene " in open, by Irish cock van Sinderen professor D. of university present.The characteristics of this carrier be introduced histidine-tagged (6 * His) with Trx A (TrxA), be beneficial to the amalgamation and expression of heterologous protein.
Lactococcus lactis ssp NZ9000 buys the institute from Dutch NIZO in the step 3, and its competent cell makes according to the milk-acid bacteria competence preparation method in " Appl Environ Microbiol, 1989 (55): 3119-3123 ".
Embodiment two: this embodiment is that the described pUC57-3-1E double digestion of in lactococcus lactis ssp, expressing in the proteic method steps two of chicken heap type Eimeria 3-1E of embodiment one is done further explanation, and the system of pUC57-3-1E double digestion is following in the step 2:
| Composition | Consumption |
| pUC57-3-1E | 14μL |
| 10×H?Buffer | 4μL |
| BamH?3 | 2μL |
| Xba?3 | 2μL |
| ddH 2O | 18μL |
The endonuclease reaction condition is 37 ℃ of reaction 3h.Other is identical with embodiment one.
Embodiment three: this embodiment is that the described expression vector pTX8048 double digestion of in lactococcus lactis ssp, expressing in the proteic method steps two of chicken heap type Eimeria 3-1E of embodiment one is done further explanation, and the system of expression vector pTX8048 double digestion is following in the step 2:
| Composition | Consumption |
| Expression vector pTX8048 | 14μL |
| 10×H?Buffer | 4μL |
| BamH?4 | 2μL |
| Xba?4 | 2μL |
| ddH 2O | 18μL |
The endonuclease reaction condition is 37 ℃ of reaction 3h.Other is identical with embodiment one.
Embodiment four: this embodiment is that further explanation is done in the one described connection of in lactococcus lactis ssp, expressing in the proteic method steps two of chicken heap type Eimeria 3-1E to embodiment, and the linked system in the step 2 is following:
| Composition | Consumption |
| The 3-1E fragment that double digestion reclaims | 6μL |
| The pTX8048 fragment that double digestion reclaims | 2μL |
| The T4DNA ligase enzyme | 1μL |
| 10 * T4DNA ligase enzyme damping fluid | 1μL |
The ligation condition is that 16 ℃ of reactions are spent the night.Other is identical with embodiment one.
Embodiment five: this embodiment is that the described PCR that in lactococcus lactis ssp, expresses in the proteic method steps three of chicken heap type Eimeria 3-1E of embodiment one is identified and do further explanation that the reaction system that PCR identifies in the step 3 is following:
The PCR reaction conditions is 94 ℃ of preparatory sex change 1min, 95 ℃ of sex change 30s, and 57.6 ℃ of 30s that anneal down, 72 ℃ extend below 1min, circulate altogether 30 times, and 72 ℃ stop extending 2min down, 4 ℃ of preservations down.Other is identical with embodiment one.
Embodiment six: this embodiment is that the embodiment one described GM17 liquid nutrient medium that contains paraxin in the proteic method steps four of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing is done further explanation, and the GM17 liquid nutrient medium that contains paraxin in the step 4 is the M17 broth culture that contains 5 μ g/mL paraxin and mass concentration 0.5% glucose.Other is identical with embodiment one.
Wherein the M17 broth culture is bought from Beijing Luqiao Technology Co., Ltd..
Embodiment:
In lactococcus lactis ssp, express the proteic method of chicken heap type Eimeria 3-1E, carry out according to the following steps:
One, will from the U.S. state-run biotechnology information center (
Www.ncbi.nlm.nih.gov) number of landing is optimized for chicken heap type Eimeria (E.acervulina) the 3-1E gene order of the EF426471 codon preference according to lactococcus lactis ssp; Obtain the 3-1E gene of synthetic; And its 5 ' end with 3 ' end introduces the recognition sequence of BamH 5 and Xba 5 respectively; Be connected and transformed into escherichia coli DH5 α competent cell screening positive transformant pUC57-3-1E afterwards with cloning vector pUC57;
Two, pUC57-3-1E and intestinal bacteria-lactic acid bacteria shuttle expression vector pTX8048 behind BamH 5 and Xba5 double digestion, are reclaimed 3-1E fragment and carrier segments respectively respectively, after the T4 dna ligase connects, make up recombinant expression vector pTX8048-3-1E;
Three, pTX8048-3-1E is transformed in the competent cell of lactococcus lactis ssp NZ9000 through electricity; Obtain containing the positive recombination lactic acid lactococcus spp NZ9000/pTX8048-3-1E of recombinant plasmid through screening; From positive recombination lactic acid lactococcus spp, extract plasmid with plasmid extraction kit; To the 3-1E gene order design primer of synthetic in the step 1, carry out PCR respectively and identify afterwards, enzyme is cut and is identified and sequencing;
Four, will identify that correct NZ9000/pTX8048-3-1E is inoculated in the GM17 liquid nutrient medium (being the M17 broth culture that contains 5 μ g/mL paraxin and mass concentration 0.5% glucose) that contains paraxin; After 30 ℃ of anaerobism overnight cultures; Transfer with 1: 50 volume ratio and to expand cultivation in the GM17 liquid nutrient medium that contains paraxin, 30 ℃ of anaerobism are cultured to OD
600Be 0.4～0.6, the nisin with 10ng/ml continues inducing culture 3h afterwards, has promptly accomplished and in lactococcus lactis ssp NZ9000, has expressed 3-1E albumen.
In the step 1 number of landing be the chicken heap type Eimeria 3-1E gene order of EF426471 shown in SEQ ID NO:1, the 3-1E gene order of synthetic is shown in SEQ ID NO:2.The 3-1E gene of synthetic is synthetic by Nanjing Jin Sirui biotech firm in the step 1.
The system of pUC57-3-1E double digestion is following in the step 2:
| Composition | Consumption |
| pUC57-3-1E | 14μL |
| 10×H?Buffer | 4μL |
| BamH?6 | 2μL |
| Xba?6 | 2μL |
| ddH 2O | 18μL |
The endonuclease reaction condition is 37 ℃ of reaction 3h.
The system of expression vector pTX8048 double digestion is following in the step 2:
| Composition | Consumption |
| Expression vector pTX8048 | 14μL |
| 10×H?Buffer | 4μL |
| BamH?6 | 2μL |
| Xba?6 | 2μL |
| ddH 2O | 18μL |
The endonuclease reaction condition is 37 ℃ of reaction 3h.
Linked system in the step 2 is following:
| Composition | Consumption |
| The 3-1E fragment that double digestion reclaims | 6μL |
| The pTX8048 fragment that double digestion reclaims | 2μL |
| The T4DNA ligase enzyme | 1μL |
| 10 * T4 dna ligase damping fluid | 1μL |
The ligation condition is that 16 ℃ of reactions are spent the night.
The reaction system that PCR identifies in the step 3 is following:
The PCR reaction conditions is 94 ℃ of preparatory sex change 1min, 95 ℃ of sex change 30s, and 57.6 ℃ of 30s that anneal down, 72 ℃ extend below 1min, circulate altogether 30 times, and 72 ℃ stop extending 2min down, 4 ℃ of preservations down.
The PCR qualification result is as shown in Figure 1; M is DNA Marker (DL2000) among the figure, is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom, and swimming lane 1,2 and 3 is PCR result; Can find out that 3-1E purpose clip size is 510bp, with the expection big or small identical.
To cut identification system following for enzyme in the step 3:
| Composition | Consumption |
| The plasmid that from positive recombination lactic acid lactococcus spp, extracts | 4μL |
| 10×H?Buffer | 1μL |
| BamH?7 | 0.5μL |
| Xba?7 | 0.5μL |
| ddH 2O | 7μL |
The endonuclease reaction condition is 37 ℃ of reaction 3h.It is as shown in Figure 2 that enzyme is cut qualification result; M is DNA Marker (DL2000) among the figure; Be 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom, swimming lane 1 and 2 is pTX8048-3-1E double digestion result, and downcutting 3-1E purpose clip size is 510bp.
Sequencing is to deliver to Nanjing Jin Sirui biotech firm to check order in the step 3, and sequencing result shows that the 3-1E gene of optimization successfully inserts among the purpose carrier pTX8048.
Claims (6)
1. in lactococcus lactis ssp, express the proteic method of chicken heap type Eimeria 3-1E for one kind, it is characterized in that in lactococcus lactis ssp, expressing the proteic method of chicken heap type Eimeria 3-1E, carry out according to the following steps:
One, chicken being piled type Eimeria 3-1E gene order is optimized according to the codon preference of lactococcus lactis ssp; Obtain the 3-1E gene of synthetic; And its 5 ' end with 3 ' end introduces the recognition sequence of BamH 1 and Xba 1 respectively; Be connected and transformed into escherichia coli DH5 α competent cell screening positive transformant pUC57-3-1E afterwards with cloning vector pUC57;
Two, with pUC57-3-1E and intestinal bacteria-lactic acid bacteria shuttle expression vector pTX8048 respectively behind BamH 1 and Xba 1 double digestion, reclaim 3-1E fragment and carrier segments respectively, connect back structure recombinant expression vector pTX8048-3-1E through the T4DNA ligase enzyme;
Three, pTX8048-3-1E is transformed in the competent cell of lactococcus lactis ssp NZ9000 through electricity; Obtain containing the positive recombination lactic acid lactococcus spp NZ9000/pTX8048-3-1E of recombinant plasmid through screening; From positive recombination lactic acid lactococcus spp, extract plasmid with plasmid extraction kit; To the 3-1E gene order design primer of synthetic in the step 1, carry out PCR respectively and identify afterwards, enzyme is cut and is identified and sequencing;
Four, NZ9000/pTX8048-3-1E is inoculated in the GM17 liquid nutrient medium that contains paraxin, after 30 ℃ of anaerobism overnight cultures, transfers with 1: 50 volume ratio and to carry out amplification cultivation in the GM17 liquid nutrient medium that contains paraxin, 30 ℃ of anaerobism are cultured to OD
600Be 0.4～0.6, the nisin with 10ng/ml continues inducing culture 3h afterwards, has promptly accomplished and in lactococcus lactis ssp NZ9000, has expressed 3-1E albumen.
2. a kind of proteic method of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing according to claim 1 is characterized in that the system of pUC57-3-1E double digestion in the step 2 is following:
The endonuclease reaction condition is 37 ℃ of reaction 3h.
3. a kind of proteic method of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing according to claim 1 is characterized in that the system of expression vector pTX8048 double digestion in the step 2 is following:
The endonuclease reaction condition is 37 ℃ of reaction 3h.
4. a kind of proteic method of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing according to claim 1 is characterized in that the linked system in the step 2 is following:
The ligation condition is that 16 ℃ of reactions are spent the night.
5. a kind of proteic method of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing according to claim 1 is characterized in that the reaction system that PCR identifies in the step 3 is following:
The PCR reaction conditions is 94 ℃ of preparatory sex change 1min, 95 ℃ of sex change 30s, and 57.6 ℃ of 30s that anneal down, 72 ℃ extend below 1min, circulate altogether 30 times, and 72 ℃ stop extending 2min down, 4 ℃ of preservations down.
6. a kind of proteic method of chicken heap type Eimeria 3-1E of in lactococcus lactis ssp, expressing according to claim 1, the GM17 liquid nutrient medium that it is characterized in that containing in the step 4 paraxin is the M17 broth culture that contains 5 μ g/mL paraxin and mass concentration 0.5% glucose.
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