CN106978431B - Lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain and function verification method thereof - Google Patents

Abstract

The invention provides a lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain and a function verification method thereof, the technical scheme is that a pair of primers is designed firstly, the primers are used for amplifying a pBpp protein expression gene containing a Usp45 lactococcus lactis secretory expression signal peptide by taking zebra fish feces flora DNA as a template, then a pMG36e plasmid is used as a vector, the plasmid is amplified in escherichia coli MC1061, and then the plasmid is introduced into lactococcus lactis MG1363 in an electrotransfer mode. The technology relies on the characteristic that lactococcus lactis colonizes intestinal tracts, can play a role in protecting the pBpp protein in a gastric acid environment, concentrates the secretion and expression places of recombinant proteins in the intestinal tract environment, realizes the in-vivo expression of the pBpp protein, and improves the absorption effect. Experiments show that the pBpp recombinant protein expression strain provided by the invention can efficiently induce the proliferation of beta cells in pancreas.

Description

Lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain and function verification method thereof

Technical Field

The invention relates to the technical field of genetic engineering, further relates to a construction technology of a recombinant protein expression system, and particularly relates to an attenuated salmonella typhimurium-mediated pBpp recombinant protein expression strain and a function verification method thereof.

Background

Diabetes is a series of metabolic disorder syndromes of sugar, protein, fat, water, electrolyte and the like caused by hypofunction of pancreatic islets, insulin resistance and the like due to the action of various pathogenic factors such as genetic factors, immune dysfunction, microbial infection and toxins thereof, free radical toxins, mental factors and the like on organisms, and is clinically characterized by hyperglycemia.

At present, the prevalence rate of type II diabetes mellitus in China is increased rapidly, according to the survey in 1979 among 30 ten thousand population of China, the prevalence rate of diabetes mellitus is 0.6%, 2.02% in 1989 and about 0.1% per year, 20 ten thousand population are generally surveyed in 1994, the prevalence rate is increased to 2.5%, and the prevalence rate of diabetes mellitus in 20-75 years old population is about 3% at present. The patients with impaired glucose tolerance have not less than 3 percent. Generally speaking, the prevalence rate of diabetes in affluent areas of China is higher than that in poor areas, cities are higher than rural areas, obesity is higher than normal weight, and the elderly are higher than those in the elderly. At present, the incidence rate of diabetes in China is about 1/1000, the average prevalence rate of diabetes over 50 years old is over 7%, the prevalence rate of diabetes under 40 years old is increased along with the increase of the age, and the peak age of a patient is 50-70 years old.

The prevalence rate of China is higher in Beijing, Liaoning, Ningxia, Gansu, Yunnan and Fujian, and the research result in 1979-1997 shows that the prevalence rate of China is the first nationwide, but Sinkiang, Guizhou and Shanxi are lower. The difference between Beijing and Liaoning with higher incidence and Guizhou and Xinjiang with the lowest incidence is up to 10 times, the urban incidence is 1-4 times higher than that in rural areas, and the investigation in Guangxi areas proves that the incidence of diabetes in sugar-producing areas is 2 times higher than that in non-sugar-producing areas.

Although various treatments can control blood glucose concentration, no cure for diabetes has been achieved at present. In the prior art, the pharmaceutical intervention is still the main means for treating diabetes, wherein GLP-1 analogues, DPPIV inhibitors and the like are widely applied. The pBpp protein (promoting beta-Cell proliferation protein) is a functional protein capable of inducing beta Cell proliferation in pancreas and further promoting insulin secretion, and has mild hypoglycemic effect and less side effect on human body because it does not directly participate in human body sugar metabolism mechanism. However, the large-scale production of the protein is a direct technical problem restricting the clinical application of the protein, wherein the acquisition of the human protein is difficult, and the drug effect and the safety of the pBpp protein from other biological sources are difficult to guarantee. In addition, although having a definite β cell proliferation-inducing effect, the pBpp protein is absorbed in a human body at a low efficiency and is easily decomposed and inactivated particularly in a gastric acid environment, and thus the efficacy is not ideal when the pBpp protein is orally administered.

Disclosure of Invention

The invention aims to provide a lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain and a function verification method thereof aiming at the technical defects in the prior art so as to solve the technical problem that a pBpp protein expression system is lacked in the prior art.

The invention also aims to solve the technical problem that when the pBpp protein is orally administered, the absorption efficiency is low and the drug effect is not obvious.

The invention also aims to solve the technical problem that the regulation effect of the pBpp protein expression strain on the blood sugar of mammals in the prior art is difficult to accurately and quantitatively investigate.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain is prepared by the following method:

1) performing PCR amplification by taking total DNA of the zebra fish fecal flora as a template and taking fragments with sequences shown as SEQ ID No.1 and SEQ ID No.2 as upstream and downstream primers respectively to obtain a DNA fragment with a sequence shown as SEQ ID No.3, namely a pBpp protein expression gene containing a Usp45 lactococcus lactis secretory expression signal peptide;

2) connecting the pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis obtained in the step 1) to a pMG36e vector to obtain a recombinant plasmid pMG36e-Usp 45-pBpp;

3) transferring the recombinant plasmid pMG36e-Usp45-pBpp obtained in the step 2) into Escherichia coli MC1061 to obtain a recombinant strain;

4) culturing the recombinant strain obtained in the step 3), extracting a recombinant plasmid pMG36e-Usp45-pBpp from the recombinant strain, introducing the recombinant plasmid pMG36e-Usp45-pBpp into competent lactococcus lactis MG1363 through an electrotransformation method, and screening out positive clones through erythromycin resistance to obtain a recombinant expression strain pMG36e-Usp45-pBpp-MG1363, namely the recombinant expression strain of pBpp protein mediated by lactococcus lactis MG 1363.

Preferably, the pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis in the step 2) is connected with the pMG36e vector by double enzyme digestion of Pst I and Hind III, and the enzyme digestion product is connected to the pMG36e vector by adopting T4 ligase after being purified.

Preferably, the electrotransformation method in the step 4) comprises the following steps: and mixing the recombinant plasmid pMG36e-Usp45-pBpp with the competent lactococcus lactis MG1363 in an electric rotating cup, and then carrying out electric rotation for 4.7ms under the conditions of voltage of 1.8kv, resistance of 200 omega and capacitance of 25 uF.

Preferably, the competent lactococcus lactis MG1363 of step 4) is prepared by:

A) inoculating frozen lactococcus lactis MG1363 into an MRS liquid culture medium, performing anaerobic static culture at 28-32 ℃ for 8-12 h, coating 80-120 mu L of bacterial liquid on an M17 solid culture medium plate, and performing anaerobic static culture at 28-32 ℃ until a single bacterial colony is formed;

B) selecting a single colony, inoculating the single colony in 3-7 ml of M17 liquid culture medium, performing anaerobic static culture at 28-32 ℃ for 8-12 h, inoculating the obtained bacterial liquid in 1.8-2.2% of volume ratio in M17 liquid culture containing 1.8-2.2% of volume fraction glycine, and performing anaerobic static culture at 28-32 ℃ until the OD value at 600nm is 0.3-0.4;

C) after ice bath is carried out for 8-12 min, centrifuging, taking out and washing the precipitate with a glycerol aqueous solution with the temperature of 0-4 ℃ and the volume fraction of 8-12%, then centrifuging, taking out and suspending the precipitate in a glycerol aqueous solution with the volume fraction of 8-12%, and carrying out ice bath for 8-12 min.

Preferably, the rotation speed of the first two centrifugations in the step C) is 5000rpm, the centrifugation time is 5min, and the rotation speed of the third centrifugation is 8000rpm, and the centrifugation time is 10 min.

A function verification method of the pBpp recombinant protein expression strain comprises the following steps:

1) taking a mouse, and continuously injecting streptozotocin at a dose of 50mg/kg per day for 5 days to obtain a type II diabetes model mouse;

2) taking a pBpp recombinant protein expression strain, and administrating the II type diabetes model mouse obtained in the step 1) in a gastric perfusion mode, wherein the dosage of each administration is 10⁸CFU/day, 1 dose every 2 days; the type II diabetes model mice were monitored for changes in blood glucose every 7 days.

In the above technical scheme, the expression gene of the pBpp protein containing the secretory expression signal peptide of Usp45 lactococcus lactis means that the product expressed by the gene fragment is the pBpp protein connected with the secretory expression signal peptide of Usp45 lactococcus lactis. The pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis is inevitably obtained by PCR amplification of primers with sequences of SEQ ID No.1 and SEQ ID No.2, namely, the primers capable of obtaining the specific gene fragments are specifically designed in a primer design link.

The invention provides a lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain and a function verification method thereof, the technical scheme is that a pair of primers is designed firstly, the primers are used for amplifying a pBpp protein expression gene containing a Usp45 lactococcus lactis secretory expression signal peptide by taking zebra fish feces flora DNA as a template, then a pMG36e plasmid is used as a vector, the plasmid is amplified in escherichia coli MC1061, and then the pBpp protein expression gene is introduced into lactococcus lactis MG1363 in an electric shock conversion mode. The technical scheme is based on the characteristic that lactococcus lactis colonizes intestinal tracts, can play a role in protecting the pBpp protein in a gastric acid environment, concentrates the secretion and expression places of the recombinant protein in the intestinal tract environment, realizes the in-vivo expression of the pBpp protein, improves the absorption effect of the pBpp protein, and lays a foundation for the proliferation and regulation effects of the pBpp protein on islet beta cells.

On the basis, in order to investigate the blood sugar reducing effect of the strain, the invention establishes a corresponding functional verification method, the method firstly utilizes streptozotocin to construct a type II diabetes model mouse, then adopts a gastric perfusion mode to carry out live bacteria administration, and analyzes the blood sugar reducing effect of the strain according to the blood sugar change condition of the mouse after administration. Experiments show that the pBpp recombinant protein expression strain provided by the invention can efficiently induce the proliferation of beta cells in pancreas, thereby having a treatment effect on diabetes caused by pancreatic beta cell injury.

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified; in the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged; in the following examples,% is by mass unless otherwise specified.

In the following examples, the e.coli MC1061, lactococcus lactis MG1363, pMG36e vector plasmids used were purchased from bio-engineering (shanghai) gmbh; the restriction enzyme, T4DNA ligase and DNA ligase used were purchased from NEB company; the LB solid medium and liquid medium used were prepared by the laboratory.

Example 1

1. Construction of pBpp recombinant protein expression Strain

PCR amplification is carried out by taking zebra fish fecal flora DNA as a template and SEQ ID No.1 and SEQ ID No.2 sequences as primers to obtain a pBpp fragment shown in SEQ ID No.3, which is named as Usp 45-pBpp.

The obtained pBpp fragment and plasmid pMG36e were digested simultaneously with Pst I and Hind III, the digested products were purified, and ligated into pMG36e (SEQ ID No.4) plasmid using T4 ligase, and the recombinant plasmid was named pMG36e-Usp 45-pBpp.

Inoculating lactococcus lactis MG1363 cryopreserved at the ultralow temperature of-80 ℃ into 5mL of MRS liquid culture medium, and performing anaerobic standing culture at the temperature of 30 ℃ overnight; coating 100 μ l of the recovered bacterial liquid on M17 plate, and performing anaerobic static culture at 30 deg.C to obtain single colony; selecting a single colony which grows well, inoculating the single colony in 5mL of M17 liquid culture medium, and performing anaerobic standing overnight culture at the temperature of 30 ℃; inoculating the above bacterial liquid into 200mL of M17 liquid culture containing 2% glycine at 2% dosage, and performing anaerobic static culture at 30 deg.C until thallus OD_600nmThe value is 0.3-0.4, and the mixture is collected for standby; ice-bath the collected thallus culture for 10min, centrifuging at 4 deg.C at 5000rpm/min for 5min, and collecting thallus; the precipitate was washed twice with ice cold 10% glycerol, each 20mL in volume; centrifuging at 4 deg.C for 10min at 8000rpm, and collecting precipitate; finally, the pellet was resuspended in 1/10 vol 10% glycerol and used after ice-bath for 10 min.

The obtained recombinant plasmid pMG36e-Usp45-pBpp was electrotransferred into lactococcus lactis MG1363 using a 0.1cm electrotransfer under the conditions set to: 1.8kv 200 omega 25uF, 4.7ms of electric conversion; screening positive clones by erythromycin resistance to obtain recombinant lactococcus lactis MG1363 which is named as pMG36e-Usp45-pBpp-MG 1363.

2. Functional verification of pBpp recombinant protein expression strain

Reagent preparation: 0.1M citric acid buffer (PH 4.5): 4.5mL of 0.1M citric acid solution (0.21014 citric acid dissolved in 10mL of distilled water) + 5.5mL of 0.1M sodium citrate solution (0.29412 sodium citrate dissolved in 10mL of distilled water); 7.5mg/mL sterile STZ solution (37.5 mg STZ dissolved in 5mL citric acid buffer, and filtered with 0.22um ultra-clean bench filter, and packaged at-20 deg.C) (care and avoid light)

The experimental process comprises the following steps:

(1) dosage and administration mode: positive group: STZ solution 50 mg/kg/day; negative group: citric acid buffer solution with the same dosage as the positive group;

the administration mode comprises the following steps: performing intraperitoneal injection;

(2) 12 healthy male mice are taken and observed for 24 hours, and the test is carried out after no abnormality exists; the mice were randomly divided into 2 groups and weighed; starving for 24 hours before the first administration, and fasting without water supply; after weighing, converting the dose of the positive group, injecting STZ medicine into the abdominal cavity of the super clean bench, after beating, normally feeding the STZ medicine into feed and sterile water for feeding, and continuously injecting the STZ medicine for 5 days at the same time; after finishing beating for 3, 7, 10, 14, 21 and 28 days, testing the blood sugar of the mice after 8 hours of fasting and the blood sugar of the mice after 2 hours of feeding by using test paper, and if the positive group is more than 11.1mmol/L, successfully molding;

(3) after the molding is successful, the stomach is perfused with pMG36e-Usp45-pBpp-MG1363 and 10 percent of stomach⁸CFU/time, and gavage frequency of 2 days, and normal diet; negative group, gavage PBS as control;

(4) detecting the blood sugar of the mice after 8h fasting on the 7 th day, the 14 th day and the 21 st day, and then feeding the mice for 2h to observe whether the blood sugar is reduced;

example 2

A lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain is prepared by the following method:

1) performing PCR amplification by taking total DNA of the zebra fish fecal flora as a template and taking fragments with sequences shown as SEQ ID No.1 and SEQ ID No.2 as upstream and downstream primers respectively to obtain a DNA fragment with a sequence shown as SEQ ID No.3, namely a pBpp protein expression gene containing a Usp45 lactococcus lactis secretory expression signal peptide;

2) connecting the pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis obtained in the step 1) to a pMG36e vector to obtain a recombinant plasmid pMG36e-Usp 45-pBpp;

3) transferring the recombinant plasmid pMG36e-Usp45-pBpp obtained in the step 2) into Escherichia coli MC1061 to obtain a recombinant strain;

4) culturing the recombinant strain obtained in the step 3), extracting a recombinant plasmid pMG36e-Usp45-pBpp from the recombinant strain, introducing the recombinant plasmid pMG36e-Usp45-pBpp into competent lactococcus lactis MG1363 through an electrotransformation method, and screening out positive clones through erythromycin resistance to obtain a recombinant expression strain pMG36e-Usp45-pBpp-MG1363, namely the recombinant expression strain of pBpp protein mediated by lactococcus lactis MG 1363.

On the basis of the technical scheme, the following conditions are met:

in the step 2), the pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis is connected with the pMG36e vector by carrying out double enzyme digestion on Pst I and Hind III, and after the enzyme digestion product is purified, the enzyme digestion product is connected to the pMG36e vector by adopting T4 ligase.

The electrotransformation method in the step 4) comprises the following steps: and mixing the recombinant plasmid pMG36e-Usp45-pBpp with the competent lactococcus lactis MG1363 in an electric rotating cup, and then carrying out electric rotation for 4.7ms under the conditions of voltage of 1.8kv, resistance of 200 omega and capacitance of 25 uF.

Step 4) said competent lactococcus lactis MG1363, prepared by the method of:

A) inoculating frozen lactococcus lactis MG1363 into an MRS liquid culture medium, performing anaerobic static culture at 28 ℃ for 8 hours, coating 80 mu L of bacterial liquid on an M17 solid culture medium plate, and performing anaerobic static culture at 28 ℃ until a single bacterial colony is formed;

B) selecting a single colony, inoculating the single colony in 3ml of M17 liquid culture medium, performing anaerobic static culture at 28 ℃ for 8 hours, inoculating the obtained bacterial liquid in 1.8% volume ratio in M17 liquid culture containing 1.8% volume fraction glycine, performing anaerobic static culture at 28 ℃ until the OD value at 600nm is 0.3;

C) after ice-bath for 8min, centrifuging, washing the precipitate with 8% glycerol aqueous solution at 0 deg.C, centrifuging, suspending the precipitate in 8% glycerol aqueous solution, and ice-bath for 8 min.

The rotating speed of the first two times of centrifugation in the step C) is 5000rpm, the centrifugation time is 5min, and the rotating speed of the third time of centrifugation is 8000rpm, and the centrifugation time is 10 min.

A function verification method of the pBpp recombinant protein expression strain comprises the following steps:

1) taking a mouse, and continuously injecting streptozotocin at a dose of 50mg/kg per day for 5 days to obtain a type II diabetes model mouse;

2) taking a pBpp recombinant protein expression strain, and administrating the II type diabetes model mouse obtained in the step 1) in a gastric perfusion mode, wherein the dosage of each administration is 10⁸CFU/day, 1 dose every 2 days; the type II diabetes model mice were monitored for changes in blood glucose every 7 days.

Example 3

A lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain is prepared by the following method:

1) performing PCR amplification by taking total DNA of the zebra fish fecal flora as a template and taking fragments with sequences shown as SEQ ID No.1 and SEQ ID No.2 as upstream and downstream primers respectively to obtain a DNA fragment with a sequence shown as SEQ ID No.3, namely a pBpp protein expression gene containing a Usp45 lactococcus lactis secretory expression signal peptide;

2) connecting the pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis obtained in the step 1) to a pMG36e vector to obtain a recombinant plasmid pMG36e-Usp 45-pBpp;

3) transferring the recombinant plasmid pMG36e-Usp45-pBpp obtained in the step 2) into Escherichia coli MC1061 to obtain a recombinant strain;

4) culturing the recombinant strain obtained in the step 3), extracting a recombinant plasmid pMG36e-Usp45-pBpp from the recombinant strain, introducing the recombinant plasmid pMG36e-Usp45-pBpp into competent lactococcus lactis MG1363 through an electrotransformation method, and screening out positive clones through erythromycin resistance to obtain a recombinant expression strain pMG36e-Usp45-pBpp-MG1363, namely the recombinant expression strain of pBpp protein mediated by lactococcus lactis MG 1363.

On the basis of the technical scheme, the following conditions are met:

step 4) said competent lactococcus lactis MG1363, prepared by the method of:

A) inoculating frozen lactococcus lactis MG1363 into an MRS liquid culture medium, performing anaerobic static culture at 32 ℃ for 12 hours, coating 120 mu L of bacterial liquid on an M17 solid culture medium plate, and performing anaerobic static culture at 32 ℃ until a single colony is formed;

B) selecting a single colony, inoculating the single colony in 7ml of M17 liquid culture medium, performing anaerobic static culture at 32 ℃ for 12h, inoculating the obtained bacterial liquid in 2.2% volume ratio in M17 liquid culture containing 2.2% volume fraction glycine, performing anaerobic static culture at 32 ℃ until the OD value at 600nm is 0.4;

C) after ice-bath for 12min, centrifuging, taking the precipitate, washing the precipitate with a glycerol aqueous solution with the temperature of 4 ℃ and the volume fraction of 12%, then centrifuging, taking the precipitate, suspending the precipitate in a glycerol aqueous solution with the volume fraction of 12%, and carrying out ice-bath for 12 min.

Example 4

A lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain is prepared by the following method:

1) performing PCR amplification by taking total DNA of the zebra fish fecal flora as a template and taking fragments with sequences shown as SEQ ID No.1 and SEQ ID No.2 as upstream and downstream primers respectively to obtain a DNA fragment with a sequence shown as SEQ ID No.3, namely a pBpp protein expression gene containing a Usp45 lactococcus lactis secretory expression signal peptide;

2) connecting the pBpp protein expression gene containing the secretory expression signal peptide of the Usp45 lactococcus lactis obtained in the step 1) to a pMG36e vector to obtain a recombinant plasmid pMG36e-Usp 45-pBpp;

3) transferring the recombinant plasmid pMG36e-Usp45-pBpp obtained in the step 2) into Escherichia coli MC1061 to obtain a recombinant strain;

4) culturing the recombinant strain obtained in the step 3), extracting a recombinant plasmid pMG36e-Usp45-pBpp from the recombinant strain, introducing the recombinant plasmid pMG36e-Usp45-pBpp into competent lactococcus lactis MG1363 through an electrotransformation method, and screening out positive clones through erythromycin resistance to obtain a recombinant expression strain pMG36e-Usp45-pBpp-MG1363, namely the recombinant expression strain of pBpp protein mediated by lactococcus lactis MG 1363.

A function verification method of the pBpp recombinant protein expression strain comprises the following steps:

1) taking a mouse, and continuously injecting streptozotocin at a dose of 50mg/kg per day for 5 days to obtain a type II diabetes model mouse;

2) taking a pBpp recombinant protein expression strain, and administrating the II type diabetes model mouse obtained in the step 1) in a gastric perfusion mode, wherein the dosage of each administration is 10⁸CFU/day, 1 dose every 2 days; the type II diabetes model mice were monitored for changes in blood glucose every 7 days.

The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of Nanchang

<120> lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain and function verification method thereof

<130> 2017

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<170> PatentIn version 3.3

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tataattata gcacggtcga tcttctatat aaaagatata ttatcttatc agtattgtca 2400

atatattcaa ggcaatctgc ctcctcatcc tcttcatcct cttcgtcttg gtagcttttt 2460

aaatatgggt cgatcgaatt cggtcctcgg gatatgataa gattaatagt tttagctatt 2520

aatctttttt tatttttatt taagaatggc ttaataaagc ggttactttg gatttttgtg 2580

agcttggact agaaaaaaac ttcacaaaat gctatactag gtaggtaaaa aaatattcgg 2640

aggaattttg aaatggcaat cgtttcagca gaaaaattcg taattcgagc tcgcccgggg 2700

atcgatcctc tagagtcgac ctgcaggcat gcaagcttgc aaagtctgaa aacgaaggtg 2760

gcagctgccg ttgaagcggc caagacagtt ggtaaaggcg acggtacaac cggtactagc 2820

gacaaaggcg gcggtcaagg taccccggcg ctacgatatt tggagttgag gttcaaagtc 2880

aaatggtact gatgaccggt aaaatttaat attttgaacc ttgcttaggc agctgacttc 2940

acattgttga gatcagctgc cttttgctta tagttcattg agtagaaacg gttctgttgc 3000

gaagtttgaa aatcaaacgc aagctcgatt ttttattaaa acgtctcaaa atcgtttctg 3060

agacgtttta gcgtttattt cgtttagtta tcggcataat cgttaaaaca ggcgttatcg 3120

tagcgtaaaa gcccttgagc gtagcgtggc tttgcagcga agatgttgtc tgttagatta 3180

tgaaagccga tgactgaatg aaataataag cgcagcgccc ttctatttcg gttggaggag 3240

gctcaaggga gtatgaggga atgaaattcc ctcatgggtt tgattttaaa aattgcttgc 3300

aattttgccg agcggtagcg ctggaaaatt tttgaaaaaa atttggaatt tggaaaaaaa 3360

tggggggaaa ggaagcgaat tttgcttccg tactacgacc ccccattaag tgccgagtgc 3420

caatttttgt gccaaaaacg ctctatccca actggctcaa gggtttaagg ggtttttcaa 3480

tcgccaacga atcgccaacg ttttcgccaa cgttttttat aaatctatat ttaagtagct 3540

ttattgttgt ttttatgatt acaaagtgat acactaactt tataaaatta tttgattgga 3600

gttttttaaa t 3611

Claims (5)

1. A preparation method of a lactococcus lactis MG 1363-mediated pBpp recombinant protein expression strain is characterized by comprising the following steps:

1) taking a DNA fragment with a sequence shown as SEQ ID No.3 as a pBpp protein expression gene containing a secretory expression signal peptide of Usp45 lactococcus lactis, and connecting the pBpp protein expression gene to a pMG36e vector to obtain a recombinant plasmid pMG36e-Usp 45-pBpp;

2) taking the recombinant plasmid pMG36e-Usp45-pBpp obtained in the step 1), and transferring the recombinant plasmid pMG36e-Usp45-pBpp into Escherichia coli E.coli MC1061 to obtain a recombinant strain;

3) culturing the recombinant strain obtained in the step 2), extracting a recombinant plasmid pMG36e-Usp45-pBpp from the recombinant strain, introducing the recombinant plasmid pMG36e-Usp45-pBpp into competent lactococcus lactis MG1363 through an electrotransformation method, and screening out positive clones through erythromycin resistance to obtain a recombinant expression strain pMG36e-Usp45-pBpp-MG1363, namely the recombinant expression strain of pBpp protein mediated by lactococcus lactis MG 1363.

2. The method of claim 1, wherein the pBpp protein expressing gene containing the signal peptide for secretion expression of Usp45 lactococcus lactis in step 1) is ligated to the pMG36e vector by digesting with Pst I and Hind III, and after purifying the digested product, the digested product is ligated to the pMG36e vector using T4 ligase.

3. The method according to claim 1, wherein the electrotransformation method of step 3) comprises the steps of: and mixing the recombinant plasmid pMG36e-Usp45-pBpp with the competent lactococcus lactis MG1363 in an electric rotating cup, and then carrying out electric rotation for 4.7ms under the conditions of voltage of 1.8kv, resistance of 200 omega and capacitance of 25 uF.

4. Method according to claim 1, characterized in that said competent lactococcus lactis MG1363 of step 3) is prepared by:

A) inoculating frozen lactococcus lactis MG1363 into an MRS liquid culture medium, performing anaerobic static culture at 28-32 ℃ for 8-12 h, coating 80-120 mu L of bacterial liquid on an M17 solid culture medium plate, and performing anaerobic static culture at 28-32 ℃ until a single bacterial colony is formed;

B) selecting a single colony, inoculating the single colony in 3-7 ml of M17 liquid culture medium, performing anaerobic static culture at 28-32 ℃ for 8-12 h, inoculating the obtained bacterial liquid in 1.8-2.2% of volume ratio in M17 liquid culture containing 1.8-2.2% of volume fraction glycine, and performing anaerobic static culture at 28-32 ℃ until the OD value at 600nm is 0.3-0.4;

C) after ice bath is carried out for 8-12 min, centrifuging, taking out and washing the precipitate with a glycerol aqueous solution with the temperature of 0-4 ℃ and the volume fraction of 8-12%, then centrifuging, taking out and suspending the precipitate in a glycerol aqueous solution with the volume fraction of 8-12%, and carrying out ice bath for 8-12 min.

5. The method according to claim 4, wherein the first two centrifugations in step C) have a rotation speed of 5000rpm and a centrifugation time of 5min, and the third centrifugation has a rotation speed of 8000rpm and a centrifugation time of 10 min.