KR101475830B1 - PROCESS FOR PRODUCING TRANSFORMATION ORIGINATED FROM LACTOBACILLUS ACIDOPHILUS COMPRISING PIG IFN-λ GENE - Google Patents

Abstract

The present invention relates to the use of Lactobacillus < RTI ID = 0.0 > acidophilus , A transformant prepared from the transformant, and a feed additive containing the transformant.

The L. acidophilus of the present invention The survival was maintained during the passage for 29 days without antibiotics, and it was dominant as a beneficial bacterium in the intestines to inhibit the proliferation of harmful bacteria, promote digestion, enhance immunity to the intestines and soften the stomach. The L. acidophilus of the present invention adhered to the intestinal mucosa The effect of excretion of pathogens and carcinogens that cause intestinal diseases was excelled.

Transgenic, feed additive, pig IFN-λ gene, Lactobacillus acidophilus

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a transformant derived from Lactobacillus acidophilus containing a Pigment AFN-gamma gene, a transformant prepared from the transformant, and a feed additive containing the transformant, -λ GENE}

The present invention relates to the use of Lactobacillus < RTI ID = 0.0 > acidophilus , A transformant prepared from the transformant, and a feed additive containing the transformant.

Lactobacillus acidophilus is a type of probiotic known to be effective in reducing abdominal pain caused by irritable bowel syndrome in humans and animals. Lactobacillus acidophilus, one of the Lactobacillus strains, is a rod-shaped lactic acid bacterium and is known to have strong acid tolerance to acid, and has a function of acting and anticancer effects, lowering of cholesterol in blood and synthesis of vitamin B group. Lactobacillus < RTI ID = 0.0 > acidophilus ) helps control many microorganisms that survive in the intestines. In general, about 400 kinds of intestinal bacterial layers exist in the intestines of normal people, among which there are beneficial bacteria such as lactic acid bacteria, but there are bacteria which are harmful to human body. Lactobacillus acidophilus is a common pathogen that causes adherence to the intestinal mucosa to cause intestinal diseases and carcinogens. It is also known as Lactobacillus acidophilus, because it inhibits the proliferation of harmful bacteria, promotes digestion, strengthens the immune system in the intestines and softens the stomach. Is excellently excreted in vitro.

Recently, a technique of isolating a strain having excellent acid production using a BCP medium from a feces of a dog and then obtaining a novel Lactobacillus acidophilus having excellent acid resistance by inoculating it into a MRS liquid medium has been disclosed (a method of culturing Lactobacillus acidophilus Culture of L actobacillus acidophilus, Republic of Korea Patent Publication No: 10-2002-0069327 (30.08.2002)}, after already extracted chromosomal DNA from Lactobacillus acidophilus (ATCC4356) to a slp promoter using PCR (P a slp) and termination sequence ( T slp A), RNA was extracted from the porcine spleen and then amplified by RT-PCR using a pig IFN-λ gene. The amplified three genes were inserted into a cloning vector (pUC19) No transformants derived from Lactobacillus acidophilus were produced.

In order to solve the above technical problems, the present invention provides a method for producing a transformant derived from Lactobacillus acidophilus containing a pig IFN-λ gene, a transformant prepared from the transformant, and a feed additive containing the transformant.

The present invention relates to a method of amplifying chromosomal DNA from Lactobacillus acidophilus (ATCC 4356), amplifying the slp A promoter (P slp A) and the termination sequence (T slp A) by PCR, and extracting the RNA from the pig spleen, -λ gene; and a step of inserting the amplified three genes in sequence into a cloning vector (pUC19) to perform recombination, and gene cloning from the recombinant pUC19 into an expression vector (pNZ123) And a method for producing a transformant derived from Lactobacillus acidophilus containing a pig IFN-lambda gene, which comprises preparing a transformant.

Among the constructs of the present invention, P slp A (promoter) using the chromosomal DNA of Lactobacillus acidophilus ATCC4356 as template,

(Forward 5'-GGC GGA ATT CGG TAA GCG GTA GGT GAA-3 '

Reverse 5'-GCG CTC TAG ATG GTC TTT TCC TCC TTG-3 '),

And T slp A (termination sequence)

(Forward 5'-GCA ACT GCA GTA ATA AGT CGT AGC ACT AAC-3 '

Reverse 5'-GGC CAA GCT TCA GAA GAT CCT ATT AGA ACT-3 ')

The transformant derived from Lactobacillus acidophilus containing the pig IFN-lambda gene, which is amplified by PCR using the primer pairs, is prepared.

In another aspect of the present invention, the Pig lambda gene, which is a template of cDNA synthesized from RNA extracted from porcine spleen,

(Pig IFN? -F: 5 'ATG CTC TAG ACT CTC TCC GAA ACA ATG AGT-3'

Pig IFN-? R: 5 'ATG CGT CGA CCA GGA TGA CAA TTA TTT TGA TG-3')

And transforming the Lactobacillus acidophilus- derived transformant containing the pig IFN-lambda gene by RT-PCR amplification using the primer pair.

Another aspect of the present invention is a feed additive for livestock containing the transformant and a transformant derived from Lactobacillus acidophilus containing a pig IFN-lambda gene produced by the method of any one of the above methods, do.

Lactobacillus know even Phil Ruth (Lactobacillus acidophilus) determine the reserves of swine TP slp A-pigIFNγ gene in the results, make an excellent effect can be long-term survival than in the field of livestock animals or humans pigs than other Lactobacillus acidophilus in the figure 19 . That is, the L. acidophilus of the present invention The survival was maintained during the passage for 29 days without antibiotics, and it was dominant as a beneficial bacterium in the intestines to inhibit the proliferation of harmful bacteria, promote digestion, enhance immunity to the intestines and soften the stomach. The L. acidophilus of the present invention adhered to the intestinal mucosa The effect of excretion of pathogens and carcinogens that cause intestinal diseases was excelled.

In the following, the details of the construction of the present invention will be described with reference to examples, test examples and the following drawings.

Example

One. Lactobacillus acidophilus ( ATCC4356 )from chromosomal DNA  extraction

Lactobacillus acidophilus (ATCC 4356) was cultured in Luria-Bertani agar medium (BD biochemical co., Sparks, USA) at 37 ° C for 24 hours (h), and then bacterial colonies were collected from which DNA was extracted. In the DNA extraction method, the cultured bacterial colonies were suspended in 100 μl of sterilized distilled water, and the bacterial colonies were incubated at 100 ° C. for 10 minutes and then at -20 ° C. for 20 minutes. The bacteria colonies were allowed to stand at room temperature . Thereafter, the mixture was centrifuged at 3,000 rpm for 1 minute, and the separated supernatant was collected and used as a template for PCR.

2. PCR amplification of the slpA promoter and termination sequence

2.1. PCR primer and condition

P slp A (promoter) and T slp A (termination sequence) with chromosomal DNA of Lactobacillus acidophilus (ATCC4356) were amplified by PCR using the following primer pairs.

PslpA primers :

Forward 5'-GGC GGA ATT CGG TAA GCG GTA GGT GAA-3 '

Reverse 5'-GCG CTC TAG ATG GTC TTT TCC TCC TTG-3 '

TslpA primers :

Forward 5'-GCA ACT GCA GTA ATA AGT CGT AGC ACT AAC-3 '

Reverse 5'-GGC CAA GCT TCA GAA GAT CCT ATT AGA ACT-3 '

The PCR reaction was pre-denaturation at 95 ° C for 5 minutes, followed by 35 cycles of reaction at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute. Finally, the reaction was further performed at 72 ° C for 7 minutes. The amplified PCR product was electrophoresed on 0.7% agarose gel for 40 minutes. After completion of the electrophoresis, a band of P slp A (275 bp) and T slp A (117 bp) Respectively. The results of the electrophoresis of PCR-amplified P slp A (promoter) and T slp A (termination sequence) genes are shown in FIG.

 2.2. P slp A and T slp A gel elution of the gene (gene)

As shown in FIG. 2, P slp A and T slp A bands were each advantageously obtained using a gel extraction kit from Qiagen. The extraction method followed the process of the manufacturer of Qiagen. The above process is briefly described. First, gels containing P slp A or T slp A band and buffer QG are mixed at a ratio of 1: 3, and the mixture is allowed to stand in a water bath at 50 ° C. for 10 minutes The gel was completely dissolved. P slp A or T slp A band and the same amount of isopropanol was added and mixed well. The mixture was transferred to a QIA quick spin column and centrifuged at 13,000 rpm for 1 minute ( The centrifugation conditions in the following procedure are the same at 13,000 rpm / 1 min). To completely separate the gel, 0.5 ml of buffer QG was further added and centrifuged. After that, 0.75 ml of buffer PE was added for washing and centrifuged. The separated bottom layer was discarded and centrifuged once more to completely remove the foreign material. The QIA Quick spin column was transferred to a new EP tube and the PCR products, P slp A and T slp A gene, were amplified using 50 μl of buffer EB.

3. RNA extraction from pig spleen

The pig's RNA was extracted using Qiagen's RNeasy Mini Kit. The extraction method followed the above manufacturer's process. A brief description follows. After splenectomy, cells were freed using a cell strainer (BD biosciences, USA) and 5 ㅧ 10 ⁶ free splenocytes were suspended in 600 μl of RLT buffer supplemented with β-mercaptoethanol And dissolved. Next, the same amount of ethanol was added and mixed well. The mixture was centrifuged in an RNeasy column for RNA adsorption. Thereafter, the RNA was washed with a washing buffer, and RNA was purified using RNase-free water.

4. Amplification of pig IFN-γ gene by RT-PCR

4.1. cDNA synthesis

CDNA was synthesized using RNA extracted from spleen according to the product manual of SuperScript ^TM First-Strand synthesis system (Invitrogen, USA). A brief description follows. 7 μl of total RNA (50 ng / μl), 1 μl of random hexamers (50 ng / μl), 1 μl of 10 mM dNTP and 1 μl of DEPC-treated water were mixed well and reacted at 65 ° C for 5 minutes. . At this time, 2 μl of 10 x RT buffer, 25 mM MgCl ₂ , 2 μl of 0.1 M DTT and 1 μl of RNase OUT recombinase inhibitor (Recombinant Ribonuclease Inhibitor) were mixed and prepared. The thus prepared mixture was mixed with a sample which had been put in ice and reacted at 25 ° C for 2 minutes. Next, 1 μl of Super Script ^™ (50 units) was added and reacted again at 25 ° C for 10 minutes. Thereafter, reaction was carried out at 42 캜 for 50 minutes and at 70 캜 for 15 minutes in that order. Finally, 1 μl of RNase H was added and reacted at 37 ° C for 20 minutes to complete cDNA synthesis.

4.2. PCR primers and their conditions

Pig IFN-γ was amplified by PCR using the synthesized cDNA as a template. The primer pairs used in the PCR are as follows.

Primer

Pig IFN? -F: 5'-ATG CTC TAG ACT CTC TCC GAA ACA ATG AGT-3 '

Pig IFNγ-R: 5'-ATG CGT CGA CCA GGA TGA CAA TTA TTT TGA TG-3 '

The PCR reaction was pre-denaturation at 95 ° C for 5 minutes, followed by 35 cycles of reaction at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute. Finally, the reaction was further performed at 72 ° C for 7 minutes. The amplified PCR product was electrophoresed in 0.7% agarose gel for 40 minutes. After completion of the electrophoresis, a Pig IFN-γ (526 bp) band was observed under UV. The electrophoresis results are shown in FIG.

4.3. Pig IFN-γ gene gel elution

The pig IFN-γ gene band as shown in FIG. 3 was advantageously obtained using Qiagen's gel extraction kit. The method of extraction is the same as the description of "2.2. P slp A and T slp A gene gel elution".

5. Three genes amplified were inserted into a cloning vector (pUC19) in turn

DH5a (a strain of E. coli) was used as a competent cell (hereinafter referred to as Competent cell) for transformation of the pUC19 vector, and the size of the pUC19 vector was 2686 bp. Competent cells were cultured using LB (BD biochemical co., Sparks, USA) medium supplemented with ampicillin.

5.1. pUC19 vector For T slp A gene  Clone ( Clone )

5.1.1. T slp Cutting A gene into restriction enzymes

T slp A was treated with restriction enzymes HindIII and PstI. The results are shown in FIG.

5.1.2. Cutting pUC19 vector with restriction enzyme

The pUP19 vector was treated with restriction enzymes HindIII and PstI. The results are shown in FIG.

5.1.3. T slp Insert A into pUC19 vector

5.1.3.1 T slp A and pUC19 vector gel elution

As shown in FIGS. 4 and 5, the T slp A gene and the pUC19 vector band were advantageously obtained using Qiagen's gel extraction kit. The method of extraction is the same as that of "2.2. P slp A and T slp A gene gel elution.

5.1.3.2 T slp A gene and pUC19 vector ligation

The free T slp A gene and pUC19 vector were ligated using T4 DNA ligase. The ligation method is as follows. 2 μl of pUC19 vector, 6 μl of T slp A gene, 2 μl of T4 DNA ligase and 10 μl of T4 ligase buffer x2 were mixed and allowed to stand overnight at 16 ° C.

5.1.3.3. T slp A gene-inserted pUC19 vector was transformed with competent cell (DH5?)

 The transformation method is briefly described as follows.

First, 20 μl of the pUC19 vector with the inserted T slp A gene and 100 μl of competent cell (DH5α) were placed in an EP tube and allowed to stand in ice for 20 minutes. Next, the sample was allowed to stand in a 42 ° C water bath for 1 minute and again on ice for 2 minutes. Then, 950 L of LB broth medium was added and cultured at 37 캜 in a shaking bath at 150 rpm / 1.5 hrs. Next, the cells were cultured in LB agar medium supplemented with amphicillin for 24 hours to confirm formation of bacterial colonies. The bacterial colonies formed were cultured for 16 hours in LB broth supplemented with ampicillin.

5.1.3.4. T slp A gene-inserted pUC19 vector was extracted from DH5α

T slp A-pUC19 vector was extracted using Plasmid DNA purification kit from Intron. The extraction method followed the above manufacturer's process. A brief description follows.

First, 3 ml of LB broth supplemented with Ampicillin, in which the Tl slp A-pUC19 vector was transfected with DH5α, was centrifuged to separate the cells. Thereafter, the cells were suspended by resuspension buffer, and the suspended cells were lysed using a lysis buffer and then neutralized by adding neutralization buffer. After centrifugation, the dissolved cell debris was discarded and only the upper layer vector was collected. The collected vector was treated with washing buffer once more, and finally the vector was purified using 50 μl of elution buffer.

5.1.3.5. T slp Confirmation of success of cloning of A gene and pUC19 vector

The results were confirmed by treatment with restriction enzymes. The results are shown in FIG.

5.2. pUC19 - T slp A vector For P slp A gene  Clone

The PslpA gene was cloned into pUC19 already containing the TslpA gene. As in the case of inserting the T slp A gene into the pUC19 vector, DH5a was used as competent cells for the pUC19 vector, and the size of the pUC19-T slp A vector was 2823 bp.

5.2.1. T slp Cutting A gene into restriction enzymes

P slp A was sequentially treated with restriction enzymes XbaI and EcoRI. The results are shown in FIG.

5.2.2. pUC19-T slp Crop a vector with a restriction enzyme

The pUC19-T slp A vector was treated in turn with XbaI and EcoRI. The results are shown in FIG.

5.2.3. P slp A of pUC19 - T slp A vector Insert into

5.2.3.1 P slp A and pUC19-T slp A vector gel elution

The P slp A gene and the T slp A-pUC19 vector band as shown in FIGS. 7 and 8 were advantageously obtained using Qiagen's gel extraction kit. The method of extraction is the same as that of "2.2. P slp A and T slp A gene gel elution.

5.2.3.2 P slp A gene and pUC19-T slp A vector ligation

The free P slp A gene and T slp A-pUC19 vector were ligated using T4 DNA ligase. The ligation method is as follows.

First, 2 μl of pUC19-T slp A vector, 6 μl of P slp A gene, 2 μl of T4 DNA ligase and 10 μl of T4 ligase buffer x2 were mixed and allowed to stand overnight at 16 ° C.

5.2.3.3. P slp Agene-inserted pUC19-T slp Transformation of Avector into DH5α

The transformation method is the same as the method described in "5.1.3.3 Transformation of pUC19 vector with T slp A gene into competent cell (DH5α)".

5.2.3.4. P slp A gene inserted pUC19-T slp A vector (pUC19-PT slp A) was extracted from DH5?

After transformation, the three bacterial colonies were formed. Extraction of pUC19-PT slp A vector from each colony was performed using Intron's plasmid DNA purification kit. The extraction method followed the above manufacturer's process.

5.2.3.5. P slp A gene and pUC19-T slp Check whether cloning A vector is successful

The results were confirmed after the same restriction enzyme treatment as the above method, and the results are shown in Fig. It was assumed that sample B was successfully cloned during the above three bacterial colonies, and only sample B was treated with restriction enzyme again. The result is shown in FIG. As a result, the process of inserting the PT slp A gene into the pUC19 vector of the present invention succeeded. Therefore, we will denote the vector as pUC19- PT slp A after the procedure below.

5.3. pUC19 - PT slp A vector For pig IFN -γ ( pIFN γ) Gene clone

Competent cells for the pUC19 vector were DH5a.

5.3.1. Cutting pIFNγ gene with restriction enzyme

Pig IFN-y gene was treated with restriction enzymes SalI and Xbal. The result is shown in FIG.

5.3.2. pUC19-PT slp Cutting Avector into Restriction Enzymes

The pUC19-PT slp A vector was treated sequentially with SalI and XbaI. The result is shown in FIG.

5.3.3. pIFN? was transfected with pUC19-PT slp Insert in A vector

5.3.3.1 pIFNγ and pUC19-PT slp A vector gel elution

PIFNγgene and pUC19-TP slp A vector band as shown in FIGS. 11 and 12 were advantageously obtained using a Qiagen gel extraction kit. The above extraction method is the same as the description of "2.2. P slp A and T slp Agene gel elution.

5.3.3.2 pIFN gamma gene and pUC19-PT slp Avectorligation

The free P slp A gene and T slp A-pUC19 vector were ligated using T4 DNA ligase. The ligation method is as follows. First, 2 μl of pUC19-PT slp A vector, 6 μl of pIFNγgene, 2 μl of T4 DNA ligase and 10 μl of T4 ligase buffer x2 were mixed and allowed to stand overnight at 16 ° C.

5.3.3.3. The pIF19 gene-inserted pUC19-PT slp A vector (pUC19-PT slp A-pIFN?) Into DH5?

 The transformation method is the same as the previous transformation method.

5.3.3.4. pUC19-PT slp A-pIFNγ was extracted from DH5α

After the transformation, three bacterial colonies were formed. Extraction of pUC19-PT slp A vector from each colony was performed using Intron's plasmid DNA purification kit. The extraction method followed the manufacturer's process.

5.3.3.5. pIFN gamma gene and pUC19-PT slp Check whether cloning of A vector is successful

The above restriction enzyme was treated to confirm the result. The results are shown in FIG.

6. pUC19 to PT slp Gene cloning of A-pigIFNγ into an expression vector (pNZ123)

Competent cells for pNZ123 vector were JM109 and pNZ123 vector was 2.8 kbp. Competent cells were cultured with LB (BD biochemical co., Sparks, USA) supplemented with chloramphenicol.

6.1. PT slp Cutting A-pigIFNγgene with restriction enzymes

The pUC19-PTslpA-pigIFN gamma gene was treated with restriction enzymes EcoRI and HindIII. The result is shown in FIG.

6.2. Cutting pNZ123 vector with restriction enzyme

The pNZ123 vector was treated in turn with restriction enzymes EcoRI and HindIII. The results are shown in FIG.

6.3. PT slp Insert A-pigIFNγgene into pNZ123 vector

6.3.1 PT slp A-pigIFNγ and pNZ123 vector gel elution

PT slp A-pigIFNγgene and pNZ123 vector band as shown in FIGS. 14 and 15 were obtained by using Qiagen's gel extraction kit. The method of extraction is the same as that of "2.2. P slp A and T slp A gene gel elution.

6.3.2 PT slp A-pigIFN gamma and pNZ123 vector ligation

The free gene and pNZ123 vector were ligated using T4 DNA ligase. The ligation method is as follows.

First, 2 μl of pUC19-PT slp A vector, 6 μl of pIFNγgene, 2 μl of T4 DNA ligase and 10 μl of T4 ligase buffer x2 were mixed and allowed to stand overnight at 16 ° C.

6.3.3. PT slp A-pigIFNγgene-inserted pNZ123 vector (pNZ123-PT slp A-pIFN?) Was transformed with JM109

 The above transfection method is the same as the previous transfection method.

6.3.4. pNZ123-PT slp A-pIFNγ was extracted from JM109

Several bacterial colonies were formed after transformation. Extraction of pNZ123-PT slp A-pIFNγvector from each colony was performed using the "quick plasmid screening" method. A brief description follows. Bacterial colonies were floated in 8 μl of protoplast buffer. After dissolving the cells in the lysis buffer, the results were confirmed by electrophoresis. In this case, if the band is above the untreated vector, the gene insert is successfully inserted into the vector, and if the band is at the same position, the vector is not inserted. As confirmed successfully.

6.3.5. PT slp Confirmation of cloning success of A-pigIFNγgene and pNZ123 vector

The results were confirmed by treatment with restriction enzymes. The result is shown in FIG.

7. TP slp A and pNZ123 in which pig IFN gamma was inserted L. acidophilus Electricity drilling conducted

The conditions for electroporation and culture medium for L. acidophilus were as follows.

* Transform pNZ123-PTslpA-pigIFNγto Lactobacillus acidophilus

Electroporation condition:

- Use 0.2-cm cuvette

- C = 25 μF

- V = 2.1 kV

- PC = infinite ohms

- Medium: MRS agar

Transformation was carried out using the above electroporation method. The result was confirmed by PCR after boiling of L. acidophilus DNA. The result is shown in FIG.

8. L. acidophilus Of pNZ123-TP slp Confirmation of A-pigIFNγgene retention

The pNZ123-TP slp A-pigIFN gamma gene in L. acidophilus was tested for the duration of subculture without antibiotics. The above-mentioned L. acidophilus was inoculated on a medium to which no antibiotic was added, and then DNA of L. acidophilus was extracted by boiling in the bacteria every day. Then, the extracted DNA was used as a template for gene cloning and the presence or absence of pNZ123-TP slp A-pigIFNγ gene was confirmed by PCR. The results showed that viability was maintained while the L. acidophilus was subcultured for 29 days, as shown in Figure 19, without antibiotic treatment.

1. L. acidophilus according to the present invention Derived pNZ123-PT slp A-pigIFN gamma gene.

Figure 2 is a photograph of the result of electrophoresis confirmation of PCR amplified P slp A (promoter) and T slp A (termination sequence) gene.

Fig. 3. Electrophoresis result. Fig. 5 shows photograph of Pig IFN-γ (526bp) band under UV.

Fig. 4. Photographs of T slp A treated with restriction enzymes HindIII and PstI.

Fig. 5. Photographs of the pUP19 vector treated with restriction enzymes HindIII and PstI.

Fig. 6. Photograph of T slp A- pUP19 treated with restriction enzyme.

Fig. 7. Photograph of the result of treating P slp A with restriction enzymes XbaI and EcoRI.

Fig. 8. Photograph of pUC19-T slp A vector treated with XbaI and EcoRI.

FIG. 9. Photographs showing the success of cloning of pUC19-T slp A vector after restriction enzyme treatment.

Figure 10. Photograph of the result of restriction enzyme treatment of sample B cloning during 3 bacterial colonies.

Figure 11. Photographs of Pig IFN-γ gene treated with restriction enzymes SalI and XbaI.

12. Photograph of pUC19-PT slp A vector treated with SalI and XbaI.

Fig. 13. Photographs showing the success of cloning of pIFNγ gene and pUC19-PT slp A vector after restriction enzyme treatment.

Figure 14. Photographs of pUC19-PTslpA-pigIFNγ gene treated with restriction enzymes EcoRI and HindIII.

Fig. 15. Photograph of pNZ123 vector treated with restriction enzymes EcoRI and HindIII.

16 is a photograph showing that the insert of pNZ123-PT slp A-pigIFNγ gene has been successfully confirmed.

Fig. 17. Photograph of the result of confirmation of cloning of PT slp A-pigIFN gamma and pNZ123 vector treated with restriction enzyme.

Fig. 18. Photograph of pNZ123-PT slp A-pigIFNγ gene obtained by boiling and extracting DNA of L. acidophilus .

19. It is confirmed that the L. acidophilus contains pNZ123-PT slp A-pigIFN gamma.

Claims (5)

Lactobacillus amplifying the slp A promoter (P slp A) and the termination sequence (T slp A) by PCR after extracting chromosomal DNA from S. aureus acidophilus (ATCC 4356); RNA extraction from pig spleen followed by amplification of pig IFN-λ gene by RT-PCR; Inserting the amplified three genes in sequence into a cloning vector (pUC19) for recombination; The recombinant pUC19 gene cloning was performed with an expression vector (pNZ123) to produce a transformant containing the pig IFN-lambda gene. Lactobacillus containing the pig IFN-lambda gene acidophilus ≪ / RTI > 2. The method according to claim 1, wherein the Lactobacillus acidophilus Derived from Lactobacillus acidophilus containing the pig IFN-λ gene, characterized in that the chromosomal DNA of ATCC4356 is used as a template and the P slp A (promoter) and T slp A (termination sequence) are amplified by PCR using the following primer pairs: ≪ / RTI > PslpA primers: Forward 5'- GGC GGA ATT CGG TAA GCG GTA GGT GAA-3 ' Reverse 5'-GCG CTC TAG ATG GTC TTT TCC TCC TTG-3 ' TslpA primers: Forward 5'-GCA ACT GCA GTA ATA AGT CGT AGC ACT AAC-3 ' Reverse 5'-GGC CAA GCT TCA GAA GAT CCT ATT AGA ACT-3 ' 2. The method according to claim 1, wherein the Pig lambda gene is subjected to RT-PCR amplification using the primer pair used in the following PCR using cDNA synthesized from the RNA extracted from porcine spleen as a template, and Lactobacillus acidophilus ≪ / RTI > Primer Pig IFN-λ F: 5 'ATG CTC TAG ACT CTC TCC GAA ACA ATG AGT-3' Pig IFN-λ R: 5 'ATG CGT CGA CCA GGA TGA CAA TTA TTT TGA TG-3' A pig IFN-lambda gene-containing Lactobacillus < RTI ID = 0.0 > acidophilus Derived transformant A feed additive for livestock containing a transformant derived from Lactobacillus acidophilus containing the pig IFN-λ gene of claim 4.

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