CN103146738A - Construction method and purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors - Google Patents
Construction method and purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors Download PDFInfo
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Abstract
The invention discloses a construction method and a purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors. Primers containing specific enzyme cutting sites are designed according to disclosed gene sequences and expression vector plasmid characteristics of the pig epidermal growth factors; pig lower jaw glandular tissues are taken as the material, a segment containing pig epidermal growth factor genes is obtained through a reverse transcription-polymerase chain reaction (RT-PCR), and the segment is connected with a lactobacillus expression vector plasmid pMG36e to obtain a recombinant plasmid pMG36e-egf, the recombinant plasmid is placed into lactobacillus acidophilus with an electrotransformation method to obtain the recombinant lactobacillus acidophilus expressing the pig epidermal growth factors, expression is performed under the induction of nisin, and correct expression of target genes is proved through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The recombinant lactobacillus acidophilus is used for preventing diarrhea occurrence, improving intestinal tract health of pigs and promoting growth performance.
Description
Technical field
The invention belongs to the animal and veterinary technical field, relate in particular to construction process and the application in promoting chitling road health thereof of a kind of restructuring Lactobacterium acidophilum of expressing pig's epidermal growth factor.
Background technology
Current, raising pigs becomes the important industry that involves the interests of the state and the people already.In Swine Production, piglet is the stage the fastest, that efficiency of feed utilization is the highest, potentiality to be exploited is maximum of growing, and the quality of feeding piglet directly affects the production achievement of the whole feeding period of pig.Yet the piglet stage is also the stage of viability, physiological function most fragile, and the piglet mortality ratio accounts for 70% of whole feeding period mortality ratio usually.Piglet survival ratio hangs down and poor growth is the subject matter that faces in present China Swine Production, and improving the feeding piglet achievement is the key that ensures the pig industry fast development.
Animal intestinal is not only the organ of digesting and assimilating of the interior maximum of animal body, is the final absorption places of all nutritive substances, is also simultaneously immune organ maximum in body, and the intestine of young pigs healthy development is the physiological foundation of Fast Growth and high-survival rate.Newborn piglet is in due to enteron aisle among Rapid development, structure and function imperfection, and digesting and assimilating with the immunization barrier function of nutrient has significant limitation, is difficult to adapt to nutrition and environmental evolution, increased the difficulty of raising.Between Piglet at Lactation Period, high-caliber, as can to promote intestinal growth and maturation somatomedin is all contained in colostrum and normal Ruzhong, as glutamine, Urogastron, insulin-like growth factor and polyamines etc., after wean, these factors disappear, to the growth generation detrimentally affect of intestinal cells growth, differentiation and cell function.The piglet ablactation is rear due to the impact that is subjected to the multiple stressors such as nutrition, physiology, environment and pathogenic micro-organism, cause intestinal mucosa atrophy, gut epithelium integrity and the gut barrier effect is destroyed and intestinal function is disorderly, cause maldigestion, diarrhoea, growth-inhibiting, even dead.According to statistics, the diarrhea of weaned piglets rate is generally 20%～30%, and rate of death caused by diarrhoea accounts for 40% of piglet general mortality rate.Therefore, promote the intestine of young pigs healthy development in production reality in the urgent need to adopting an effective measure, improve and digest and assimilate and the immunization barrier function.
Pig's epidermal growth factor is as one of higher somatomedin of pig Ruzhong content, and it has impact of crucial importance to the newborn piglet gastrointestinal development.Early weaning is the important means that shortens the sow breeding cycle, and the ablactation stress that early weaning causes is the major cause that affects the early-weaned piglets production performance, can alleviate ablactation stress by add Urogastron in feed.although pig's epidermal growth factor relies on its unique physicochemical property and significant physiological action, has vast potential for future development as fodder additives, but the pig's epidermal growth factor of producing at present is all obtain from milk product or utilize escherichia expression system to express gained, these two kinds of approach all have certain limitation, the amount of separating pig's epidermal growth factor from the Ruzhong is limited, being difficult to satisfying the market needs, and the pig's epidermal growth factor that utilizes escherichia expression system to express, because the colibacillary limitation of Host Strains can not directly be added use as feed, exist the difficult problem of separation and purification, increased the interpolation cost, being difficult to satisfying the market needs,
Given this present invention is take generally regarded as safe microorganisms lactobacillus acidophilus as carrier bacterium, and structure can be expressed the restructuring Lactobacterium acidophilum of pig's epidermal growth factor, and with this Lactobacterium acidophilum of recombinating as fodder additives.This carrier bacterium belongs to gram-positive microorganism, cell walls does not contain endotoxic toxic component, and has a growth that promotes the animal digestive tract probiotic bacterium, suppress the breeding of harmful bacteria, improve intestinal environment, strengthen the multiple effects such as Immune Function In Animals, be applied in Production of Livestock and Poultry as probiotic feed additive.And expressed pig's epidermal growth factor can promote to damage the reparation of gut epithelium, reaches the purpose of prevention diarrhoea.Therefore develop piglet with the recombinant probiotics of expressing the intestinal mucosa Urogastron, have broad application prospects in feed industry and pig industry.It is blank that domestic research in this field at present still belongs to.
Summary of the invention
The objective of the invention is the intestinal tract injury that causes for present stress of baby pigs caused ablaction and can not effectively repair, prior art is not enough and insurmountable present situation, construction process and the purposes of a kind of Lactobacterium acidophilum of recombinating are provided, research and develop a kind of restructuring Lactobacterium acidophilum that can promote that the intestine of young pigs mucosa injury is repaired.Restructuring Lactobacterium acidophilum of the present invention had both had Lactobacterium acidophilum to the maintenance function of intestine microenvironment, can bring into play again recombinant human epidermal growth factor to the repairing effect of chitling road damage, reach the diarrhoea that the intestinal tract injury that causes because of various factors for the treatment of piglet causes then, increase the survival rate of piglet, solve the bottleneck problem in the pig-breeding process.
Purpose of the present invention can realize by following technical proposals:
A kind of construction process of expressing the restructuring Lactobacterium acidophilum of pig's epidermal growth factor comprises the following steps:
(1) acquisition of pig's epidermal growth factor gene: extract pig lower jaw gland RNA, obtain cDNA through RT-PCR, take this cDNA as template, synthetic upstream and downstream primer P1 and the P2 that contains Sac I and Xba I restriction enzyme site of design, upstream and downstream primer P1 and P2 have respectively the sequence of pig egf gene (Gene ID:X59516.1), adopt PCR method amplification pig egf gene fragment.The egf goal gene fragment that increases is connected with cloning vector pMD18-T carrier, obtain recombinant plasmid, change in escherichia coli DH5a, through the amicillin resistance screening, the picking positive colony, and cut by enzyme and identify and the sequencing analysis checking, verify correct plasmid called after pMD18T-egf, the recombinant plasmid sequencing shows that this plasmid has the gene order of pig egf.
(2) structure of recombinant expression plasmid pMG36e-egf: lactic acid bacteria expression vectors pMG36e and cloned plasmids pMD18T-egf are carried out double digestion with restriction enzyme Sac I and Xba I, purifying reclaims linearizing pMG36e carrier segments and the egf goal gene fragment of double digestion, and linearizing carrier segments is connected with the goal gene fragment, transform in the escherichia coli DH5a competent cell, by the erythromycin resistance screening, the picking positive colony, extract recombinant plasmid, after enzyme is cut and is identified correctly, called after pMG36e-egf, it has the gene order of pig egf.
(3) structure of restructuring Lactobacterium acidophilum: adopt electric method for transformation that recombinant plasmid pMG36e-egf is transformed in Lactobacterium acidophilum NZ6092 competent cell, utilize the erythromycin resistance to screen, obtain recombinant bacterium, called after: NZ6092-egf.
(4) abduction delivering of pig's epidermal growth factor in Lactobacterium acidophilum: constructed restructuring Lactobacterium acidophilum NZ6092-egf is inoculated in the MRS liquid nutrient medium that contains 100ug/mL erythromycin, carry out abduction delivering, grope its abduction delivering condition, and expression product is carried out SDS-page and Western Blot analysis.Abduction delivering.Obtain size and be the protein band of 6Kda left and right.
(5) the anti-diarrhoea effect of gene recombination bacterium: with the constructed gene recombination Lactobacterium acidophilum piglet of feeding, observe the effect of its control grice diarrhoea.
A kind of purposes of expressing the recombinant probiotics of pig's epidermal growth factor gene, the application of the recombinant probiotics of described expression pig's epidermal growth factor gene is by adding in drinking-water or feed, promote the pig gi tract healthy, the generation of control grice diarrhoea improves growth performance.
The advantage that the present invention has is:
The recombinant probiotics of the express cell factor egf that the present invention is constructed has advantages of that traditional probiotic bacterium is incomparable.The existing Lactobacterium acidophilum of this recombinant probiotics improve intestinal environment, promote the advantage of growth of animal; Expressed cytokine egf has again the effect of repairing the enteron aisle loss.Can be used as a kind of novel fodder additives.
Description of drawings
Fig. 1 is the RT-PCR amplified production electrophorogram of pig egf gene
Fig. 2 is cloning vector pMD18T-egf restriction enzyme digestion and electrophoresis figure
Fig. 3 is expression vector pMG36e-egf restriction enzyme digestion and electrophoresis figure
Fig. 4 is that the western-blot of the egf of abduction delivering analyzes
Embodiment
Embodiment
Referring to accompanying drawing 1-Fig. 4, the restructuring Lactobacterium acidophilum of expression pig's epidermal growth factor of the present invention adopts following methods to build: according to the gene order of published pig's epidermal growth factor (egf) and the characteristics of expression vector plasmid, design contains the primer of special restriction enzyme site; Take pig lower jaw gland as material, extract mRNA, it is that template is carried out pcr amplification that reverse transcription obtains cDNA, acquisition contains the egf gene fragment, and it is connected with expression vector plasmid pMG36e, obtain recombinant plasmid pMG36e-egf, by electric method for transformation, this recombinant plasmid electricity is transformed in the Lactobacterium acidophilum competent cell, express.And with the Lactobacterium acidophilum feeding animals of constructed Recombinant Swine Urogastron, observe the effect of its control diarrhoea.Required experiment material of the present invention and embodiment are as follows:
One) experiment material
(1) goal gene sequence
The gene order of pig's epidermal growth factor EGF comes from NCBI Genebank.
(2) carrier
Nisin inducible expression carrier pMG36e is available from sea, Hunan profit bio tech ltd.
The pMD18-T cloning vector is available from the Takara bio tech ltd.
(3) bacterial strain
Escherichia coli DH5a is available from the Takara bio tech ltd, and recipient bacterium Lactobacterium acidophilum NZ6092 is available from Chinese agriculture microbial strains preservation center.
Two) embodiment
1, the acquisition of pig's epidermal growth factor EGF gene
Take the gene order (GeneID:X59516.1) of pig's epidermal growth factor of report as template, the upstream and downstream primer P1:5-G that contains Sac I and Xba I restriction enzyme site is synthesized in design
GAGCTCCGAATAGTTACTCTGAAT-3(Sac I), as SEQID NO.2 and P2:5-TA
TCTAGACGCAGCTCCCACCATTT-3(Xba I), as SEQID NO.3; Extract pig lower jaw gland RNA, obtain cDNA through reverse transcription, take this cDNA as template, by the PCR method pig egf gene fragment that increases, the PCR product reclaims the PCR product after detected through gel electrophoresis.With the egf goal gene fragment that reclaims of increase with after cloning vector pMD18-T carrier is connected, change in escherichia coli DH5a, screen by amicillin resistance, obtain recombinant plasmid, recombinant plasmid Sac I and Xba I double digestion, enzyme is cut product through detected through gel electrophoresis, and enzyme is cut and identified correct rear recombinant plasmid called after pMD18T-egf.Simultaneously recombinant plasmid is sent to Beijing Losec bio tech ltd and carries out sequencing.
2, the structure of the pMG36e of recombinant expression vector
Lactic acid bacteria expression vectors pMG36e and cloned plasmids pMD18T-egf are carried out double digestion with restriction enzyme Sac I and Xba I, the double digestion product is after gel electrophoresis analysis, purifying reclaims linearizing pMG36e carrier segments and the egf goal gene fragment of double digestion, and linearizing carrier segments is connected with the goal gene fragment, connecting product transforms in the escherichia coli DH5a competent cell, by the erythromycin resistance screening, the picking positive colony, extract plasmid, recombinant plasmid Sac I and Xba I double digestion, enzyme is cut product through detected through gel electrophoresis.Recombinant plasmid after identifying correctly, called after pMG36e-egf, it has the gene order of pig egf.
3, express the structure of the restructuring Lactobacterium acidophilum of pig's epidermal growth factor
Adopt electric method for transformation that recombinant plasmid pMG36e-egf is transformed in Lactobacterium acidophilum NZ6092 competent cell, the electric shock condition is voltage 2.5KV, resistance 400 Ω, electric capacity 25 μ F.Utilize the erythromycin resistance to screen, process PCR identifies, enzyme is cut and identified and rear recombinant bacterium called after: the NZ6092-egf of order-checking (Beijing AudioCodes bio tech ltd) checking.
4, abduction delivering and SDS, the Western Blot of pig's epidermal growth factor in Lactococcus lactis analyzes
Picking recombinant bacterium NZ6092-egf is inoculated in the MRS liquid nutrient medium that contains 100 μ g/mL erythromycin, 30 ℃ of standing overnight incubation.Culture is transferred with 3% ratio and is contained in erythromycin MRS nutrient solution in 500mL, is cultured to OD
600=0.5～0.6 o'clock, add inductor nisin, making its final concentration is 5ng/mL, continues to cultivate 3-5 hour.The recombinant bacterium cracking obtains correction by SDS-PAGE and Western blot analytical proof goal gene.Recombinant bacterium is after nisin induces, and one specific proteins band appears in the place in the 6Kda left and right, with the in the same size of expection.Gel is through thin layer scanning analysis demonstration, and target protein accounts for 8% of recipient bacterium total protein.
5, express the anti-diarrhoea effect of the restructuring Lactobacterium acidophilum of pig's epidermal growth factor
(contain restructuring Lactobacterium acidophilum 10,300,000,000/kg) is mixed and made into restructuring Lactobacterium acidophilum fodder additives with silicon-dioxide and carries out the weanling pig test restructuring Lactobacterium acidophilum bacterium liquid of the expression pig's epidermal growth factor that builds with the present invention under 37 ℃ according to envelope-bulk to weight ratio 1:50.48 of the DLY piglets of 6.23 ± 0.17kg, 21 ages in days wean are selected in test, are divided at random two groups, three repetitions of every component (hurdle), 8, every hurdle.Two groups of basal diets are consistent, and test group is added 0.05% restructuring Lactobacterium acidophilum fodder additives, and the blank group does not add restructuring Lactobacterium acidophilum fodder additives.
Three) experiment effect
1, the RT-PCR of pig's epidermal growth factor EGF gene amplification
Extract pig lower jaw gland RNA, adopt RT-PCR method amplification pig egf gene fragment, the PCR product obtains through detected through gel electrophoresis the purpose band that size is about 150bp, conforms to expected results.Result as shown in Figure 1.
2, the enzyme of cloned plasmids pMD18T-egf is cut evaluation
Cloning vector Sac I and Xba I double digestion, enzyme is cut product through detected through gel electrophoresis, and result shows that enzyme cuts out carrier segments and the big or small goal gene fragment that is about 150bp of big or small 2800bp left and right, and enzyme is cut result and is shown that the cloned plasmids structure is correct.Identify correct rear recombinant plasmid called after pMD18T-egf.Enzyme is cut result as shown in Figure 2.
3, the sequencing of pig's epidermal growth factor egf
Enzyme is cut identified that correct cloning vector is sent to Beijing AudioCodes bio tech ltd and carries out sequence and record, result shows that this plasmid has the gene order of pig egf, and sequence is as shown in SEQ ID NO.1.
4, the enzyme of expression plasmid pMG36e-egf is cut evaluation
Expression plasmid Sac I and Xba I double digestion, enzyme is cut product through detected through gel electrophoresis, and result shows that enzyme cuts out carrier segments and the big or small goal gene fragment that is about 150bp of big or small 3600bp left and right, and enzyme is cut result and is shown that the expression plasmid structure is correct.Identify correct rear recombinant expression plasmid called after pMG36e-egf.Enzyme is cut result as shown in Figure 3.
5, abduction delivering and SDS, the Western Blot of pig's epidermal growth factor in Lactococcus lactis analyzes
The Lactobacterium acidophilum of Recombinant Swine EGF obtains through abduction delivering the target protein that size is about 6KDa, obtains correction through SDS-PAGE and Western blot analytical proof goal gene.Result as shown in Figure 4.
6, express the anti-diarrhoea effect of the restructuring Lactobacterium acidophilum of pig's epidermal growth factor
The restructuring Lactobacterium acidophilum bacterium liquid of the expression pig's epidermal growth factor that builds with the present invention carries out experimentation on animals, observes it to the impact of Growth Performance of Weaning Piglets, raises and the results are shown in Table 1.
Table 1 adds the recombinant probiotics of expression pig's epidermal growth factor gene to the impact of Growth Performance of Weaning Piglets
Annotate: the female different persons of colleague's shoulder marking-up represent significant difference (p＜0.05).
Result shows, uses the recombinant probiotics of expression pig's epidermal growth factor gene of the present invention, compares with the blank group, can significantly improve 21 age in days weanling pig day weight gains 21.2%; Reduce material anharmonic ratio 10.4%; Reduce diarrhea rate 57.6%.
Claims (2)
1. construction process of expressing the recombinant probiotics of pig's epidermal growth factor gene is characterized in that comprising the following steps:
(1) acquisition of pig's epidermal growth factor gene: extract pig lower jaw gland RNA, obtain cDNA through RT-PCR, take this cDNA as template, synthetic upstream and downstream primer P1 and the P2 that contains Sac I and Xba I restriction enzyme site of design, upstream and downstream primer P1 and P2 have respectively the sequence of pig egf gene (Gene ID:X59516.1), adopt PCR method amplification pig egf gene fragment.The egf goal gene fragment that increases is connected with cloning vector pMD18-T carrier, obtain recombinant plasmid, change in escherichia coli DH5a, through the amicillin resistance screening, the picking positive colony, and cut by enzyme and identify and the sequencing analysis checking, verify correct plasmid called after pMD18T-egf, the recombinant plasmid sequencing shows that this plasmid has the gene order of pig egf.
(2) structure of recombinant expression vector pMG36e-egf: lactic acid bacteria expression vectors pMG36e and cloned plasmids pMD18T-egf are carried out double digestion with restriction enzyme Sac I and Xba I, purifying reclaims linearizing pMG36e carrier segments and the egf goal gene fragment of double digestion, and linearizing carrier segments is connected with the goal gene fragment, transform in the escherichia coli DH5a competent cell, by the erythromycin resistance screening, the picking positive colony, extract recombinant plasmid, after enzyme is cut and is identified correctly, called after pMG36e-egf, it has the gene order of pig egf.
(3) express the structure of the restructuring Lactobacterium acidophilum of pig's epidermal growth factor: adopt electric method for transformation that recombinant plasmid pMG36e-egf is transformed in Lactobacterium acidophilum NZ6092 competent cell, utilize the erythromycin resistance to screen, obtain recombinant bacterium, called after: NZ6092-egf.
(4) abduction delivering and SDS, the Western Blot of pig's epidermal growth factor in Lactobacterium acidophilum analyzes: picking recombinant bacterium NZ6092-egf is inoculated in the MRS liquid nutrient medium that contains 100ug/mL erythromycin, abduction delivering.Obtain size and be the protein band of 6Kda left and right.
2. a kind of purposes of expressing the recombinant probiotics of pig's epidermal growth factor gene according to claim 1, it is characterized in that: the recombinant probiotics of described expression pig's epidermal growth factor gene is by adding in drinking-water or feed, promote the pig gi tract healthy, the generation of control grice diarrhoea improves growth performance.
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Cited By (7)
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| CN103710351A (en) * | 2013-12-23 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Recombination lactic acid bacteria including epidermal growth factor and application thereof |
| CN104031962A (en) * | 2014-05-05 | 2014-09-10 | 四川农业大学 | Optimized pEGF gene engineering Lactococcus lactis fermentation medium |
| CN105327338A (en) * | 2015-11-23 | 2016-02-17 | 东北农业大学 | Medicine for treating ulcerative colitis and preparation method of medicine |
| CN107325998A (en) * | 2017-06-20 | 2017-11-07 | 江西嘉博生物工程有限公司 | A kind of Recombinant Lactococcus lactis and construction method for expressing pig's epidermal growth factor gene |
| CN110044881A (en) * | 2019-04-04 | 2019-07-23 | 大连大学 | A kind of detection kit and its application method of enzymatic assays creatinine |
| CN110607306A (en) * | 2019-10-15 | 2019-12-24 | 赛法特（长沙）生物技术有限公司 | Expression method of recombinant porcine epidermal growth factor |
| CN113845584A (en) * | 2021-11-08 | 2021-12-28 | 江苏三仪生物工程有限公司 | Preparation method of recombinant avian epidermal growth factor |
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| CN103710351A (en) * | 2013-12-23 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Recombination lactic acid bacteria including epidermal growth factor and application thereof |
| CN103710351B (en) * | 2013-12-23 | 2015-12-09 | 江苏众红生物工程创药研究院有限公司 | Urogastron recombinant lactic acid bacteria and application thereof |
| CN104031962A (en) * | 2014-05-05 | 2014-09-10 | 四川农业大学 | Optimized pEGF gene engineering Lactococcus lactis fermentation medium |
| CN105327338A (en) * | 2015-11-23 | 2016-02-17 | 东北农业大学 | Medicine for treating ulcerative colitis and preparation method of medicine |
| CN105327338B (en) * | 2015-11-23 | 2018-11-23 | 东北农业大学 | A kind of drug and preparation method thereof for treating ulcerative colitis |
| CN107325998A (en) * | 2017-06-20 | 2017-11-07 | 江西嘉博生物工程有限公司 | A kind of Recombinant Lactococcus lactis and construction method for expressing pig's epidermal growth factor gene |
| CN110044881A (en) * | 2019-04-04 | 2019-07-23 | 大连大学 | A kind of detection kit and its application method of enzymatic assays creatinine |
| CN110044881B (en) * | 2019-04-04 | 2021-10-12 | 大连大学 | Detection kit for measuring creatinine by enzyme method and use method thereof |
| CN110607306A (en) * | 2019-10-15 | 2019-12-24 | 赛法特（长沙）生物技术有限公司 | Expression method of recombinant porcine epidermal growth factor |
| CN113845584A (en) * | 2021-11-08 | 2021-12-28 | 江苏三仪生物工程有限公司 | Preparation method of recombinant avian epidermal growth factor |
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