CN107043769A - A kind of gene engineering method for the yield for improving lactein plnJ - Google Patents

Abstract

The present invention provides a kind of gene engineering method for the yield for improving lactein plnJ, by construction recombination plasmid, and external albumen is carried out substantial amounts of production by the characteristics of transfecting engineering bacteria, utilizing works bacterium high efficient expression.Lactein plnJ gene engineering method can be effectively improved by establishing, vivoexpression lactein plnJ, and demonstrates lactein plnJ and still have higher broad-spectrum antibacterial function.

Description

A kind of gene engineering method for the yield for improving lactein plnJ

Technical field

The invention belongs to biological technical field, and in particular to a kind of genetic engineering side of raising lactein plnJ yield Method.

Background technology

Antibiotic is widely applied as livestock and poultry antibacterial medicines and growth accelerator in Animal husbandry production.But it is long-term Using antibiotic, the prevention effect of Animal diseases is not only influenceed；Also it can develop immunity to drugs and remain, cause serious livestock products dirty Dye, the humans and animals that can even induce having produce canceration and distortion, jeopardize the health and safety of the mankind.Therefore, January 1 in 2006 Day rises, and European Union completely forbids food animal and uses antibiotic feed additive for promoting growth.Completely forbid antibiotic dynamic as food Thing feed additive for promoting growth, the increasingly most attention to cause the whole world.However, European Union is due to completely forbidding antibiotic feed Additive also brings some problems, the increase of such as enterobacterial diseases to animal productiong.In order to reverse the decline of animal health, only Largely it can be treated using therapeutic antibiotics.Simultaneously as the increase of intestinal bacteriosis also increases animal food The chance polluted by intestinal cell is for example salmonella-polluted.Therefore, livestock birds health can be improved by developing one kind, improve production performance and The product of the energy substitute antibiotics of safety non-toxic turns into a focus of Animal nutrition research.

Lactein is that have suppression by what Ribosome biogenesis mechanism was produced in metabolic process by some lactobacillus bacterias The polypeptide or Precursor Peptide of bacterium activity.Many studies have shown that, such as bacterial strain, condition of culture in fermentation process（Optimum growth temperature, training Support time, optimum pH, liquid amount etc.）And medium component（Such as carbon source, nitrogen source, phosphate）Have to bacteriocin yield Important influence.Lactein plnJ is important one kind in the bacteriocin of the secretion of lactic acid bacteria.Due to wild type lactic acid bacteria its Secretion lactein plnJ yield is similarly subjected to the influence of many factors, therefore, explores a kind of effective raising lactein The method of plnJ yield has great importance for large-scale production lactein plnJ.

Gene engineering method transfects engineering bacteria, the spy of utilizing works bacterium high efficient expression mainly by construction recombination plasmid Point, substantial amounts of production is carried out by external albumen.Therefore, by research, we, which establish, can effectively improve lactein PlnJ gene engineering method, vivoexpression lactein plnJ, and demonstrate lactein plnJ still have it is higher Broad-spectrum antibacterial function.

The content of the invention

It is an object of the invention to provide a kind of gene engineering method for the yield for improving lactein plnJ.

To achieve the above object, the present invention is adopted the following technical scheme that：

The design and synthesis of primer：With reference to GeneBank lactic acid bacterias（GeneBank:X94434）PlnJ genes nucleotides sequence Row, a pair of specific primers, plnJ sense primers F are designed using Primer Premier6： 5’- GCGGATCCATGACTGTGAACAAAATGAT-3 ', plnJ anti-sense primer R：5’- ATGTCGACTTAACGACGGATTGCTCTGC-3 ', serves the raw work bioengineering share in sea limited by designed primer sequence Company synthesizes.

A kind of gene engineering method for the yield for improving lactein plnJ, comprises the following steps：

（1）Design a pair of plnJ specific primers, plnJ sense primers F： 5’- GCGGATCCATGACTGTGAACAAAATGAT-3 ', plnJ anti-sense primer R：5’- ATGTCGACTTAACGACGGATTGCTCTGC-3’；

（2）Using the sense primer F and anti-sense primer R of plnJ genes as amplimer, lactic acid bacteria DNA is template, to plnJ bases Because entering performing PCR amplification, pcr amplification product is identified with agarose gel electrophoresis；

（3）After agarose gel electrophoresis identification PCR primer, cut purpose band and purified；By plnJ products after purification with Plasmid pET-32a (+) carries out double digestion；The PCR purpose fragments and carrier segments of digestion is taken to be attached reaction；After connection Recombinant plasmid, conversion obtains the recombinant protein for being capable of induced expression in vitro to BL21 competent cells.

The advantage of the invention is that：

（1）Using technique for gene engineering in-vitro recombination expression lactein plnJ, the technology is convenient and simple, by expanding culture work Journey bacterium can just mass produce lactein plnJ.

（2）In-vitro recombination expression lactein plnJ still have broad-spectrum antibacterial function, and with it is safe and nontoxic, without pair The advantages of effect, noresidue, feed addictive can be used as on animal productiong.

Brief description of the drawings

Fig. 1 PCR amplifications.Wherein M：DNA molecular mass standards；1：The product of plnJ gene magnifications.

Fig. 2 PCR qualification results.Wherein M：DNA Marker；1：PlnJ pcr amplification product；2：The PCR of T7 primers expands Increase production thing：3:The PCR amplifications of T7 sense primers and plnJ anti-sense primers；4：T7 anti-sense primers and plnJ sense primers PCR Product.

Fig. 3 digestion qualification results.M1、M2：DNA Marker；1：PET-32a (+)-plnJ recombinant plasmids of non-digestion； 2：PET-32a (+)-plnJ recombinant plasmidsBamH ISingle endonuclease digestion；3：The restructuring of pET-32a (+)-plnJ recombinant plasmidsSal I Single endonuclease digestion；4：PET-32a (+)-plnJ recombinant plasmidsBamH I+Sal IDouble digestion.

The optimum results of the optimal inducing temperatures of Fig. 4 IPTG.Wherein M：Protein standard；1：Blank group is induced Group；2~6：Respectively 16 DEG C, 25 DEG C, 31 DEG C, 37 DEG C, pET-32a (+)-plnJ recombinant plasmids induced under 43 DEG C of gradient temperatures Bacterium group.

The optimum results of the optimal induction times of Fig. 5 IPTG.Wherein M：Protein standard；1：Blank group is induced Group；2~6：PET-32a (+)-plnJ recombinant plasmid bacterium induced under 0 h, 4 h, 8 h, 12 h after respectively inducing, 16h gradients Group.

The optimum results of the optimal induced concentrations of Fig. 6 IPTG.Wherein M：Protein standard；1：Blank group is induced Group；2~9：Respectively IPTG induced concentrations are 0 mmoL/L, 0.2 mmoL/L, 0.4 mmoL/L, 0.6 mmoL/L, 0.8 PET-32a (+)-plnJ recombinant plasmid bacterium induced under the mmoL/L gradients of mmoL/L, 1.0 mmoL/L, 1.2 mmoL/L, 1.5 Group.

The Ni-NTA affinitive layer purification results of Fig. 7 recombinant proteins.Wherein M:Protein standard； 1：It is pure Protein solution 2~9 before changing：Respectively 40 mM, 80mM, 120 mM, 160 mM, 200 mM, 240 mM, 280 mM and 320 mM Deng the purification result of imidazole elution.

Fig. 8 Western-blot qualification results.Wherein 11：PET-32a (+)-plnJ groups；2：PET-32a (+) group；3： Blank bacterium group.

Embodiment

1 materials and methods

1.1 material

1.1.1 bacterial strain and expression vector

Bacterial strain：FQ plants of the lactic acid bacteria for being separated and being preserved by this University Of Agriculture and Forestry In Fujian animal science institute Histology and Embryology laboratory； The Host Strains of expression：E.coli DH5 α competent cells, E.coli BL 21 (DE3) competent cell（Purchased from the full formula in Beijing Golden biotech firm）；Expression vector：pET-32a(+)（Purchased from Promega Beijing Bioisystech Co., Ltd）1.

1.1.2 main agents

Bacterial genomes DNA extraction kit（DP302）, plasmid is small carries middle amount kit（DP106-02）（Purchased from Beijing Tiangeng Biochemical technology Co., Ltd）；The quick QIAquick Gel Extraction Kits of Ago-Gel DNA (CW2302)（It is century biological skill purchased from Beijing health Art company）；T4 ligases（Purchased from Promega companies）；BamH IQuickCut restriction enzymes,Sal I QuickCut is limited Enzyme,（Purchased from the precious bioengineering Co., Ltd in Dalian）；TransTaq^TM DNA Polymerase High Fidelity(HiFi) (AP131-11), ProteinIsoTM Ni-NTA Resin protein purification posts（DP101-02）, BCA determination of protein concentration reagents Box, horseradish peroxidase（HRP）The sheep anti mouse of mark and anti-His label proteins primary antibody（Purchased from Beijing Quan Shi King Companies）； Blue Plus IV Protein Marker（The kDa of 10kDa~180）；SuperSignal West Pico chemical luminous substrates （Purchased from Thermo Fischer Scient Inc.）；PAGE gel reagent preparation box（AR0138）, IPTG Glycosides (IPTG)；Remaining conventional reagent is purchased from traditional Chinese medicines company with medicine.

1.2 test method

1.2.1 the design and synthesis of primer

With reference to GeneBank lactic acid bacterias（GeneBank:X94434）PlnJ genes nucleotide sequence, using Primer Premier6 designs a pair of specific primers, plnJ sense primers F： 5’- GCGGATCCATGACTGTGAACAAAATGAT- 3 ', plnJ anti-sense primer R：5’- ATGTCGACDesigned primer sequence is served sea by TTAACGACGGATTGCTCTGC-3 ' Sheng Gong bioengineering limited company synthesizes.

1.2.2 the amplification of plnJ genes

1.2.2.1 the extraction of lactic acid bacteria STb gene

The DNA of lactic acid bacteria is extracted according to bacterial genomes DNA extraction kit specification.

1.2.2.2 PCR is expanded

Using the sense primer F and anti-sense primer R of plnJ genes as amplimer, lactic acid bacteria DNA is template, and plnJ genes are entered Performing PCR is expanded, and pcr amplification product is identified with agarose gel electrophoresis, specific PCR reaction systems and reaction condition（It see the table below 1）.

The PCR amplification system table of table 1 and reaction condition

1.2.3 the structure of recombinant expression plasmid and identification

1.2.3.1 the purifying of pcr amplification product

After agarose gel electrophoresis identification PCR primer, cut purpose band and purified, detailed step enters with reference to conventional method OK.

1.2.3.2 plasmid pET-32a (+) extraction

The plasmid pET-32a (+) that laboratory is frozen is recovered, and the 10mL LB containing ammonia benzyl is inoculated into by 1% inoculum concentration In meat soup, in the constant-temperature table for being placed in 37 DEG C, 200rpm overnight incubations carry middle amount kit extraction plasmid using plasmid is small.

1.2.3.2 the double digestion of purpose fragment and expression vector

PlnJ products after purification are subjected to double digestion with extracting obtained plasmid pET-32a (+).Plasmid pET-32a (+) is used 100uL double digestion system, plnJ after purification uses 60uL double digestion system.After double digestion reaction terminates, glue is returned again The purpose fragment and carrier segments of purifying digestion are received in case being attached reaction.

1.2.3.3 coupled reaction

The PCR purpose fragments plnJ and carrier segments of the digestion of glue reclaim purifying again are taken, enters row agarose gel electrophoresis estimation The two concentration, then using purpose fragment and carrier molar ratio as 7:1 is attached reaction, mole with calculation method of physical volume see T4 Ligase specification（Promega companies）, linked system and reaction condition（Refer to table 2 below）：

The coupled reaction system of table 2 and condition

1.2.3.4 recombinant plasmid transformed

By the recombinant plasmid after connection, conversion is expanded to DH5 α competent cells, and operating procedure is as follows：

（1）50 μ L competent cell DH5 α are added in connection product, 25min is placed on ice；

（2）After ice bath is completed, 42 DEG C of s of heat shock 45, and be immediately placed on and place 2 min on ice；

（3）250 μ L are added to balance to the LB meat soups of room temperature（It is not added with Amp+）, 200 r/min, 37 DEG C of h of oscillation incubation culture 1；

（4）200 μ L bacterium solutions are taken uniformly to be applied to the solid culture plate containing LB（Plus Amp+）, it is inverted in 37 DEG C of constant incubators In, incubated overnight（To obtain more clone, 4000 r/min centrifuge 1 min, discard part supernatant, retain 100~150 μ L, gently plays even suspension thalline, takes whole bacterium solution coated plates）.

1.2.3.5 PCR identifies that positive recombinant and recombinant plasmid are extracted

The monoclonal bacterium colony of white is selected into 10 μ L sterilized water, piping and druming is mixed, performing PCR identification is entered by template of this mixed liquor Positive recombinant plasmid or with the T7 universal primers on carrier（Table 3）Expanded.Same colony inoculation is trained in liquid LB simultaneously Support base（Containing Amp）, 200 r/min, 37 DEG C of shaken cultivations stay overnight.The matter that middle amount kit extracts the PCR positives is put forward using plasmid is small Grain DNA.

The T7 primer sequence tables of table 3

1.2.3.6 the digestion identification of recombinant expression plasmid and sequencing

Extract the positive recombinant plasmid of PCR identifications and carry out double digestion identification, specific digestion system and condition are with reference to conventional method. Agarose gel electrophoresis identifies PCR and endonuclease reaction product, and qualification result meets expected recombinant plasmid and is sent to marine growth work Journey limited company is sequenced and the correct recombinant expression plasmid of sequencing result is named as into pET-32a (+)-plnJ.

1.2.4 the analysis of lactein plnJ gene orders

Analysis sequencing result is compared using NCBI websites nucleotide sequence；Simultaneously genetic homology is carried out with Mega biosoftwares Analysis and the amino acid sequence analysis derived.

1.2.5 induced expressions of recombinant plasmid pET-32a (+)-plnJ in E.coli BL21 (DE3)

Recombinant plasmid pET-32a (+)-plnJ is converted into E.coli BL21 (DE3) competent cell, method is same 1.2.3.4 described, picking white monoclonal colony inoculation is in 1 mL LB liquid medium in flat board（Containing Amp）, put 200 It is about D600 nm=1.0 to be cultivated in r/min, 37 DEG C of shaking table to concentration, then expands culture, with 1：100 ratio is inoculated in LB fluid nutrient mediums（Containing Amp）In, 200 r/min are cultivated in 37 DEG C of shaking table, are about D600 nm=1.0 to concentration, are divided into Two parts, portion adds IPTG and expressed to final concentration of 1 mmoL/L inducible proteins, and another is added without IPTG, continues to be placed in shaking table 12 h of middle culture, while setting pET-32a (+) empty carrier bacterium induction group and blank induction group.Take 500 μ L flora suspension, 8000 R/min, 4 DEG C of 5 min of centrifugation, collects flora, is resuspended with 100 μ LPBS buffer solutions, 6 × SDS-PAGE is added according to dilution ratio Sample-loading buffer, vibration is mixed after boiling 5min, cooled on ice 5min in boiling water, and in 4 DEG C, 12000 r/min centrifuge 5min, Supernatant solution is taken to carry out protein SDS-PAGE electrophoretic analysis.

1.2.6 SDS-PAGE electrophoretic analysis

It is 15% SDS-PAGE electrophoretic separation, SDS-PAGE to draw 10 μ L above sample proteins lysates respectively and carry out gum concentration Gel is configured（Such as table 4 below）, concrete operation method and collocation method reference《Fine works molecular biology experiment guide（5th edition）. After electrophoretic separation terminates, taking-up colloid, which is placed in 0.1% (W/V) Coomassie brilliant G-250 staining solution, dyes 45 min, decolourizes Eluted in solution, taken pictures after background is decolourized totally, carry out the Preliminary Determination of protein content.

The PAGE gel of table 4 is with tabulation

1.2.7 protein induced expression system optimization

1.2.7.1 the optimization of the optimal inducing temperatures of IPTG

The positive colony flora of preservation is recovered, with 1:100 ratio is inoculated in LB culture mediums（Containing Amp）, 200 r/min, 37 When being cultivated in DEG C shaking table to D600 nm=1, adding IPTG makes its final concentration of 1.0 mmoL/L, is respectively placed in different temperature ladders Induced expression is carried out under the conditions of 16 DEG C, 25 DEG C, 31 DEG C, 37 DEG C, 43 DEG C of degree, takes 10 μ L protein samples to carry out SDS-PAGE electrophoresis point From it is determined that optimal inducing temperature.

1.2.7.2 the optimization of the optimal induction times of IPTG

Under the conditions of optimal inducing temperature, it is ensured that under the final concentration of 1.0 mmoL/L condition of culture of IPTG is constant, respectively in difference Point in time sampling：0h, 4 h, 8 h, 12 h, 16 h, take 10 μ L above protein samples to carry out SDS-PAGE electrophoretic analysis, it is determined that Optimal induction time.

1.2.7.3 the optimization of the optimal induced concentrations of IPTG

On the basis of the optimal inducing temperature and incubation time that optimization is obtained, the IPTG derivants of different final concentrations are separately added into：0 mmoL/L、0.2 mmoL/L、0.4 mmoL/L、0.6 mmoL/L、0.8 mmoL/L、1.0 mmoL/L、1.2 mmoL/L、1.5 MmoL/L is induced, and is taken 10 μ L above protein samples to carry out SDS-PAGE electrophoretic analysis, is finally determined optimal induced concentration.

1.2.8 the purifying of albumen and determination of protein concentration

1.2.8.1 Ni-NTA affinitive layer purifications of albumen

Mass propgation flora to concentration is D600 nm=1 in 200 r/min, 37 DEG C of shaking tables, adds IPTG derivants, 8000 r/ Min centrifuges 5 min, collects bacterial sediment, and PBS is washed 3 times, and brotein equilibrium buffer solution is hanged again with 1/10 volume, Ultrasonic wave crushes thalline completely under ice bath environment, and 12000 r/min centrifuge 5 min, draws supernatant and is used as sample to be purified.Foundation ProteinIsoTM Ni-NTA Resin operating methods carry out separation and purification of protein, the collection liquid that various concentrations imidazoles is eluted SDS-PAGE electrophoretic analysis is carried out, the optimal purifying concentration of imidazoles is determined.ProteinIsoTM Ni-NTA Resin are marked to His The process that label albumen is isolated and purified is generally included：Fill the steps such as post, balance, loading, washing, elution, regeneration.

1.2.8.2 the concentration mensuration of albumen after purification

With BCA method protein quantification kits, detected with ELIASA and draw standard curve, the pET-32a (+) that calculating is purified- PlnJ protein concentrations.

1.2.9 the Western blot identifications of recombinant protein

After recombinant protein pET-32a (+)-plnJ after purification are separated by electrophoresis through SDS-PAGE, it is immunized using transfer groove Trace, immuning hybridization is carried out followed by primary antibody, secondary antibody, is finally developed the color luminous and is taken pictures.

2 results and analysis

The PCR amplifications of 2.1 plnJ genes

Using lactic acid bacteria genomic DNA as template, the sense primer F and anti-sense primer R of plnJ genes are specificity amplification primer, Performing PCR amplification is entered with the reaction condition optimized, amplified production is identified through nucleic acid electrophoresis, it is seen that a size is 168bp sizes Specific band（As shown in Figure 1）, it is consistent with expected clip size, thus proves that the primer of design can be to lactic acid bacteria PlnJ genes carry out specific amplification.

The qualification result of 2.2 recombinant expression plasmids

Select using white monoclonal bacterium as template, performing PCR identification entered with the sense primer F and anti-sense primer R of plnJ genes respectively, 184bp specific band is amplified, with carrier universal primer T7 sense primer F and anti-sense primer R, can be amplified The purpose band of 867bp sizes（As shown in Figure 2）.

Digestion qualification result is illustrated in fig. 3 shown below, and the visible size of the product of recombinant plasmid single endonuclease digestion is about 6000 in figure Bp or so linear plasmid band, double digestion is visible to have more an about 184bp band, is consistent with expected size, it was demonstrated that Successful clone is on expression vector pET-32a (+) by lactein gene plnJ, and the sequencing result of recombinant plasmid is further Prove mistake of the successful structure without frameshit of plasmid.

The induced expression result of recombinant protein

2.5.1 the optimization of the optimal inducing temperatures of IPTG

Under conditions of final concentration of 1.0 mmoL/L of IPTG, 12 h of induction are carried out to pET-32a (+)-plnJ recombinant plasmids bacterium, The expression quantity of this albumen induced at a temperature of 16 DEG C, 25 DEG C, 31 DEG C, 37 DEG C, 43 DEG C is analyzed, is as a result illustrated in fig. 4 shown below, egg Expression quantity all becomes larger in 16~37 DEG C of thermograde in vain, and expression quantity all reaches maximum, 37 DEG C under conditions of 37 DEG C Expressing quantity is all gradually decreased afterwards, thus, it can be known that the optimal inducing temperature of pET-32a (+)-plnJ albumen is 37 DEG C.

2.5.2 the optimization of the optimal induction times of IPTG

Under conditions of 37 DEG C, final concentration of 1.0 mmoL/L of IPTG, pET-32a (+)-plnJ recombinant plasmids bacterium difference is analyzed Expressing quantity change under the different induction time such as 0h, 4h, 8h, 12h, 16h.As a result it is illustrated in fig. 5 shown below, albumen is being lured When leading 4 h, all start express express target protein, and the expression quantity of destination protein is gradually stepped up with the passage of time, until induction 12 Maximum is reached after h, hereafter, the expression quantity of albumen gradually tends to balance.It follows that under this condition, IPTG optimal induction Time is 12 h.

2.5.3 the optimization of the optimal induced concentrations of IPTG

The expression of pET-32a (+)-plnJ recombinant plasmids bacterium albumen in the final concentration of 0mmoL/L of IPTG seldom exists, and concentration exists When between 0.2~1.2mmoL/L gradient, the expression quantity of albumen has gradually incremental trend, is reached in 1.2mmoL/L concentration Maximum, no longer incremental during 1.5mmoL/L, synthesis result can determine that pET-32a (+)-plnJ recombinant plasmids bacterium under 37 DEG C of environment 12h is cultivated, its optimal IPTG induced concentration is 1.2mmoL/L（As shown in Figure 6）.

The Ni-NTA affinitive layer purification results of 2.6 albumen

Bacterium solution after sound is broken all uses various concentrations respectively by Ni-NTA affinity columns（40mmoL/L, 80mmoL/L, 120mmoL/L, 160mmoL/L, 200mmoL/L, 240mmoL/L, 280mmoL/L, 320mmoL/L）Imidazoles dilution carry out Washing purifying, and SDS-PAGE analyses are carried out, as a result show（Such as Fig. 7）, the protein band obtained after elution is more and more single One, illustrate that nonspecific protein impurities have been significantly reduced, but in different imidazole concentrations, obtained protein concentration and purity are not Equally, with the eluent efficiency highest of 280mM imidazole concentration.

2.7 Western-blot qualification results

As a result show（As shown in Figure 8）, learnt in being analyzed for the Western blot of His Tag label proteins, pET-32a (+)-plnJ recombinant plasmid bacterium groups and pET-32a (+) empty carrier bacterium group are about respectively 26.0 ku in size and 20 ku occur one The specific band of bar.

Antibacterial result of 2.8 pET-32a (+)-plnJ recombination lactic acids rhzomorphs to four kinds of indicator bacterias

Fungistatic effect of pET-32a (+)-plnJ recombination lactic acids rhzomorph to four kinds of indicator bacterias（As shown in table 5）, pET-32a (+)- PlnJ recombination lactic acid rhzomorphs concentration be 0.10mg/mL when to staphylococcus aureus, Escherichia coli, salmonella and Pasteurella With fungistatic effect, it is seen then that pET-32a (+)-plnJ recombination lactic acid rhzomorphs have the bacteriostatic activity of wide spectrum.

Size of pET-32a (+)-plnJ recombination lactic acids rhzomorph of table 5 to indicator bacteria inhibition zone（mm）

Presently preferred embodiments of the present invention is the foregoing is only, all equivalent changes done according to scope of the present invention patent are with repairing Decorations, should all belong to the covering scope of the present invention.

SEQUENCE LISTING

<110>University Of Agriculture and Forestry In Fujian

<120>A kind of gene engineering method for the yield for improving lactein plnJ

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Claims (2)

1. a kind of primer for the yield for improving lactein plnJ, it is characterised in that：The primer is：PlnJ sense primers F： 5’- GCGGATCCATGACTGTGAACAAAATGAT-3 ', plnJ anti-sense primer R：5’- ATGTCGACTTAACGACGGATTGCTCTGC-3’。

2. a kind of gene engineering method for the yield for improving lactein plnJ, it is characterised in that：Described method includes as follows Step：

(1) a pair of plnJ specific primers, plnJ sense primers F are designed： 5’- GCGGATCCATGACTGTGAACAAAATGAT-3 ', plnJ anti-sense primer R：5’- ATGTCGACTTAACGACGGATTGCTCTGC-3’；

（2）Using the sense primer F and anti-sense primer R of plnJ genes as amplimer, lactic acid bacteria DNA is template, to plnJ bases Because entering performing PCR amplification, pcr amplification product is identified with agarose gel electrophoresis；

（3）After agarose gel electrophoresis identification PCR primer, cut purpose band and purified；By plnJ products after purification with Plasmid pET-32a (+) carries out double digestion；The PCR purpose fragments and carrier segments of digestion is taken to be attached reaction；After connection Recombinant plasmid, conversion obtains the recombinant protein for being capable of induced expression in vitro to BL21 competent cells.