KR100622137B1 - Novel Transgenic Lactic Acid Bacteria Producing Foreign Proteins (UCCM-10355) and Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a novel transgenic lactic acid bacterium that efficiently produces foreign proteins and a method for producing the same, wherein a transgenic lactic acid bacterium is prepared by inserting a T7 RNA polymerase gene into a gram positive bacterium lactic acid genome using a homologous insertion method. Gram-positive bacteria were constructed by constructing a plasmid vector equipped with a T7 RNA polymerase promoter and an S-layer signal sequence capable of recognizing T7 RNA polymerase, and then transforming the transformed lactic acid bacterium into which the T7 RNA polymerase gene was inserted. Using lactic acid bacteria has an excellent effect to efficiently produce the desired foreign protein.

Transgenic lactic acid bacteria, Lactobacillus plantarum, T7 RNA polymerase, S layer signal sequence.

Description

New transformed lactic acid bacteria (UCCM-10355) producing foreign protein and manufacturing method thereof {NOVEL TRANSFORMED LACTOBACILLUS PRODUCING FOREIGN PROTEIN AND THE METHOD FOR THE PREPARATION OF IT}             

Figure 1 shows a schematic diagram of the lactic acid bacteria insertion vector pLT7I inserted T7 RNA polymerase gene.

Figure 2 shows a photograph showing the PCR amplification results for genomic DNA of lactic acid bacteria transformed with pLT7I

Figure 3 shows a photograph showing the result of the Sudden blot analysis for genomic DNA of lactic acid bacteria transformed with pLT7I.

Figure 4 is a picture diagram showing a PCR amplification result of the S layer signal sequence obtained from the genomic DNA of Lactobacillus brevis (Lactobacillus brevis).

5 shows a schematic diagram of the plasmid vector p123 (1-4) Endo of the present invention equipped with a T7 RNA polymerase promoter and an S layer signal sequence.

Figure 6 shows the cellulase of the final transformed lactic acid bacteria (KCCM-10355) of the present invention transformed transgenic lactic acid bacteria inserted with the T7 RNA polymerase gene with a recombinant plasmid vector equipped with a T7 RNA polymerase promoter and S layer signal sequence. The photograph shows the activity.

Figure 7 shows the overall expression schematic of the transformed lactic acid bacteria of the present invention.

The present invention relates to a novel transgenic lactic acid bacterium that efficiently expresses a foreign protein and a method for producing the same. More specifically, the T7 RNA polymerase gene that efficiently produces a foreign protein in a gram-negative bacterium is transferred to a gram-positive bacterium. A recombinant lactic acid bacterium is inserted to prepare a transformed lactic acid bacterium, and a recombinant plasmid vector capable of producing foreign protein by recognizing the T7 RNA polymerase expressed in the transformed lactic acid bacterium is constructed. It relates to a transgenic lactic acid bacteria and a method for producing the same that efficiently produce foreign proteins.

Recently, many studies have been conducted to produce foreign proteins in lactic acid bacteria, but due to various characteristics of lactic acid bacteria, development of a system for efficiently producing foreign proteins such as this is still insufficient. Currently, vector systems capable of efficiently producing foreign proteins from gram-negative or gram-positive bacteria include the T7 RNA polymerase system with great success in E. coli BL21 (DE3), which has already been commercialized. Many researchers use it as a system for mass production of foreign proteins in E. coli, a Gram-negative bacterium. However, this system is only available to Gram-negative bacteria such as E. coli and has not yet been used on Gram-positive bacteria such as lactic acid bacteria.

Meanwhile, a vector system for producing foreign proteins in Gram-positive bacteria includes a system using a signal sequence of S-layer protein (hereinafter, referred to as S-layer signal sequence) present in the cell membrane of Gram-positive bacteria. Some studies have reported the use of this system to express foreign proteins in lactic acid bacteria (Gene 1997. 186: 255-262, Appl. Microbiol. Biotechnol. 1999. 51: 71-78). .

The inventors of the present invention devised in view of the above-mentioned point to introduce a T7 RNA polymerase system for producing foreign proteins efficiently from gram negative bacteria into the gram positive bacteria lactic acid bacteria genome to produce a transgenic lactic acid bacteria, The present invention was completed by constructing a recombinant plasmid capable of producing a foreign protein by recognizing T7 RNA polymerase expressed in, and transforming the transformed lactic acid bacterium to prepare a final transformed lactic acid bacterium (KCCM-10355). .

Accordingly, an object of the present invention is to provide a transgenic lactic acid bacterium capable of efficiently producing a desired foreign protein using a gram positive bacterium lactic acid bacterium as a host, and a method for producing the same.

The object of the present invention is to prepare an insertion vector for inserting the T7 RNA polymerase gene into the lactic acid bacterium genome, and to insert the insertion vector onto the desired genome of the lactic acid bacteria to produce a transgenic lactic acid bacterium, After constructing a recombinant plasmid vector for lactic acid bacteria equipped with a T7 RNA polymerase promoter that recognizes the T7 RNA polymerase expressed in, the plasmid vector was transformed back into the transgenic lactic acid bacteria into which the T7 RNA polymerase gene was inserted. This was accomplished by developing a final transgenic lactic acid bacterium (KCCM-10355) that efficiently produces proteins.

The present invention provides a lactic acid bacterium insertion vector pLT7I prepared by inserting a T7 RNA polymerase gene into a pGEM-T vector and inserting a lac promoter into the front of the T7 RNA polymerase gene.

In another aspect, the present invention provides a lactic acid bacterium insertion vector pLT7I characterized in that it contains a chloramphenicol antibiotic site for confirming transformation in the lactic acid bacterium insertion vector pLT7I.

The present invention also provides a recombinant plasmid vector p123 (1-4) Endo prepared by ligation of the S-layer signal sequence to the pGEM-T vector and inserting a T7 RNA polymerase promoter.

In addition, the present invention inserts the T7 RNA polymerase gene into the pGEM-T vector and the lactobacillus insertion vector pLT7I prepared by inserting the lac promoter in front of the T7 RNA polymerase gene is inserted into the lactic acid bacterium genome to prepare a transgenic lactic acid bacteria Transformed lactic acid bacteria (KCCM) prepared by ligation of the S-layer signal sequence to the pGEM-T vector and the recombinant plasmid vector p123 (1-4) Endo prepared by inserting the T7 RNA polymerase promoter into the transformed lactic acid bacterium (KCCM). -10355).

In addition, the present invention inserts the T7 RNA polymerase gene into the pGEM-T vector and the lactobacillus insertion vector pLT7I prepared by inserting the lac promoter in front of the T7 RNA polymerase gene is inserted into the lactic acid bacterium genome to prepare a transgenic lactic acid bacteria Transgenic lactic acid bacterium, characterized in that the recombinant plasmid vector p123 (1-4) Endo prepared by ligation of the S layer signal sequence to the pGEM-T vector and the insertion of the T7 RNA polymerase promoter was transformed into the transgenic lactic acid bacterium. It provides a method of manufacturing (KCCM-10355).

Hereinafter, the present invention will be described in detail.

Insertion of the T7 RNA polymerase gene into the lactic acid bacteria genome is performed as follows.

First, the step of inserting the T7 RNA polymerase gene into the pGEM-T vector will be described. Extract genomic DNA of E. coli BL21 (DE3), perform PCR amplification using primers T7-N1 / T7-C1, and ligation the PCR product to the pGEM-T vector. (blue / white selection) selects colonies with inserts inserted. The oligo sequence of the synthesized primer is as follows, Bgl II is inserted into T7-N1, and the restriction enzyme site of Xba I is inserted into T7-C1.

T7-N1: AAA AGATCT GATTTACTAACTGGAAGAGGC

           Bgl ii

T7-C1: TTT TCTAGA GGATCCCGAGTCGTATTGATTT

                  Xba I

Second, the step of inserting the lac promoter into the pGEM-T vector will be described. The lac promoter present in the lactic acid bacterium vector pNZ3004 is subjected to PCR amplification using the primer lac N1 / lac C1, and the resulting PCR product is ligated to the pGEM-T vector, and colonies inserted with the inserts are selected. The lac promoter is intended to express the T7 RNA polymerase gene and is inserted at the front of the T7 RNA polymerase gene so that the T7 RNA polymerase gene is affected by the lac promoter. The oligo sequence of the synthesized primer is as follows. BamH I is inserted into lac N1 and the restriction enzyme site of Xba I is inserted into lac C1.

lac N1: TTG GGATCC TTTGCAAAATGTTCAAGATAACG

                   BamH I

lac C1: TTG TCTAGA TGATGAAATAAGAACAGAGG

                   Xba I

Third, the step of inserting the lac promoter (pGEM-LT7) before the T7 RNA polymerase gene will be described. pGEM-T7 is cut with Bgl II enzyme and pGEM-lac with BamH I, and the fragment is ligated with pGEM-T7. That is, the BamH I site of the Lac promoter and the Bgl II site of T7 are ligated. As a result, pGEM-LT7 is produced. Here, in order to cut this pGEM-LT7 with Xba I and insert the Hind III site into the fragment, the Xba I / Hind III linker was ligated with the Xba I site of the fragment, and then cut again with Hind III to the both ends of the fragment. Obtain a fragment with the Hind III site.

Fourth, the step (pGEM-PR10 / 20) of preparing a site where homologous recombination occurs with genomic DNA of Lactobacillus plantarum is described. The site where homologous recombination can occur in the genome of Lactobacillus plantarum is found in the pGIP008 vector, followed by PCR amplification to obtain the product. The primer sequence is as follows.

PR 10: TTGC TCTAGA GCTTGCTCAATCAATCACCG

                   Xba I

PR 20: CCCCG CTCGAG GCCGCCGCTGCTGCAGA

                    Xho I

Fifth, a step of inserting an antibiotic site for confirming transformation is described. After transformation, an antibiotic site is inserted into the vector to select a transformant. The primer was prepared based on the sequence of the chloramphenicol (Cm) site of the pNZ123 vector, followed by PCR amplification to clone the Cm site and to the Sal I / Sac I site of the pGEM-PR10 / 20 vector of step 4. Insert it.

Sixth, the step of constructing the final lactic acid bacterium insertion vector (pLT7I) will be described. Since the Hind III site of the vector prepared in step 5 exists at the center site of the sequence where homologous recombination can occur, the first step is to cut the vector of step 5 into Hind III and in the third step pGEM-LT7 vector, Fragments containing Hind III sites at both ends obtained by cutting with III were gel-eluted and ligated to Hind III sites of the vector prepared in step 5 to finally construct the lactic acid bacterium insertion vector pLT7I.

Seventh, the steps of confirming whether the T7 RNA polymerase gene is inserted into the Lactobacillus plantarum genome will be described. After extracting genomic DNA of the transgenic lactic acid bacteria, PCR amplification is performed using the primers T7-N1 / T7-C1 used in the first step. As a result, it can be confirmed that the 2.7kb T7 RNA polymerase gene was inserted into the genome of the transgenic lactic acid bacteria. In addition, it can be confirmed that the T7 RNA polymerase gene was inserted by Southern blotting.

The method for constructing a recombinant lactic acid bacterium expression vector equipped with a T7 RNA polymerase promoter and an S layer signal sequence is performed as follows.

First, the cloning of the S layer signal sequence from genomic DNA of Lactobacillus plantarum is described. In order to clone the S layer signal sequence region known as a gene sequence that effectively secretes foreign proteins in lactic acid bacteria, the following primers were prepared and PCR amplification was performed to obtain three fragments.

PS (1-2): TT GAATTC ACCCAGCCAGTACCAGAAGC

                   EcoR I

PS (1-3): TT GGATCC AACGACAATCAGAGCGTAA

                   BamH I

PS (1-4): TT GGATCC TAACTTCGGTTATACTATTCTTG

                   BamH I

PS2-1: CGTA TCTAGA AGCTGAAGCAGTCGTTG

                   Xba I

Secondly, the step of ligation of the fragment of the first step in front of the gene from which the signal sequence and the promoter to be expressed is removed will be described. 5 'of the desired gene is inserted into the Xba I site and 3' into the Xho I site, followed by PCR amplification to obtain the product. Thereafter, the product is cut into Xba I and Xho I, ligated with the fragment of the first step, and then PCR amplification is performed again to obtain a new fragment ligated with the signal sequence. This fragment is inserted into the pGEM-T vector, transformed, and an insert-inserted colony is selected. The plasmid is then extracted, cut with EcoR I / Xho I or BamH I / Xho I and gel eluted to obtain the fragments.

Third, the step of ligation of the T7 RNA polymerase promoter will be described. The pET-21 vector with the T7 RNA polymerase promoter is cut with EcoR I / Xho I or BamH I / Xho I and gel eluted to ligation with the fragment of the second step. After confirming the insert inserted colonies, primers ligated with the T7 RNA polymerase promoter, that is, primers based on the T7 RNA polymerase promoter site present in the pET21 vector, are prepared as follows and PCR products are obtained using the same. .

LPEN-2: TT GAATTC TAGGCGCCAGCAACCGCACC

                  EcoR I

LPEC-2: TT GTCGAC TTAATGCGCCGCTACAGGG

                  Sal I

Fourth, the step of ligation of the fragment of the third step to the lactic acid bacteria vector will be described. PNZ123, a shuttle vector of E. coli and lactic acid bacteria, was cut into EcoR I and Sal I, and then ligated with the EcoR I / Sal I fragment of the third step to transform E. coli JM 101. Thereafter, insert-inserted colonies are selected, and plasmids are extracted from the transformed E. coli to transform the lactic acid bacteria into which the T7 RNA polymerase gene is inserted. The lactic acid bacteria transformed by the above method were deposited with the Korean spawn association on February 20, 2002. (Accession No .: KCCM-10355)

Fifth, the step of confirming the foreign protein expression of the transformed lactic acid bacteria is described. Congo red test using the cellulase gene as a reporter gene is performed to confirm that the transgenic lactic acid bacteria (KCCM-10355) expresses cellulase. As a result, it can be confirmed that the cellulase activity of the transgenic lactic acid bacteria of the present invention is high.

Hereinafter, specific examples of the present invention will be described with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Insertion of T7 RNA Polymerase Gene into Lactic Acid Bacteria Genome

First step: inserting the T7 RNA polymerase gene into the pGEM-T vector

Extract genomic DNA of E. coli BL21 (DE3), perform PCR amplification using primers T7-N1 / T7-C1, and ligation the PCR product to the pGEM-T vector. Colonies with inserts were selected by (blue / white selection). The oligo sequence of the synthesized primers is as follows, Bgl II in T7-N1 and Xba I restriction enzyme sites were inserted in T7-C1.

T7-N1: AAA AGATCT GATTTACTAACTGGAAGAGGC

           Bgl ii

T7-C1: TTT TCTAGA GGATCCCGAGTCGTATTGATTT

                  Xba I

Step 2: inserting the lac promoter into the pGEM-T vector

The lac promoter present in the lactic acid bacterium vector pNZ3004 was subjected to PCR amplification using the primer lac N1 / lac C1, and the resulting PCR product was ligated to the pGEM-T vector, and colonies with inserts were selected. The lac promoter is intended to express the T7 RNA polymerase gene and is inserted at the front of the T7 RNA polymerase gene so that the T7 RNA polymerase gene is affected by the lac promoter. The oligo sequence of the synthesized primers was as follows. BamH I was inserted into lac N1 and the restriction enzyme site of Xba I was inserted into lac C1.

lac N1: TTG GGATCC TTTGCAAAATGTTCAAGATAACG

                   BamH I

lac C1: TTG TCTAGA TGATGAAATAAGAACAGAGG

                   Xba I

Step 3: inserting the lac promoter in front of the T7 RNA polymerase gene (pGEM-LT7)

pGEM-T7 was cut with Bgl II enzyme and pGEM-lac with BamH I, and the fragment was ligated with pGEM-T7. That is, the BamH I site of the Lac promoter and the Bgl II site of T7 were ligated. As a result, pGEM-LT7 was produced. Here, in order to cut this pGEM-LT7 with Xba I and insert the Hind III site into the fragment, the Xba I / Hind III linker was ligated with the Xba I site of the fragment, and then cut again with Hind III to the both ends of the fragment. Fragments with Hind III sites were obtained.

Fourth Step: Lactobacillus Planta Room To prepare a site for homologous recombination with genomic DNA of human (pGEM-PR10 / 20)

The site where homologous recombination could occur in the genome of Lactobacillus plantarum was found in the pGIP008 vector, and then PCR amplification was performed to obtain the product. Primer sequences were as follows.

PR 10: TTGC TCTAGA GCTTGCTCAATCAATCACCG

                   Xba I

PR 20: CCCCG CTCGAG GCCGCCGCTGCTGCAGA

                    Xho I

Step 5: inserting antibiotic sites to confirm transformation

Antibiotic sites were inserted into the vector to select transformants after transformation. The primer was prepared based on the sequence of the chloramphenicol (Cm) site of the pNZ123 vector, followed by PCR amplification to clone the Cm site and to the Sal I / Sac I site of the pGEM-PR10 / 20 vector of step 4. Inserted.

Step 6: construct the final lactic acid bacteria insertion vector (pLT7I)

Since the Hind III site of the vector prepared in step 5 exists at the center site of the sequence where homologous recombination can occur, the first step is to cut the vector of step 5 into Hind III and in the third step pGEM-LT7 vector, Fragments containing Hind III sites at both ends obtained by cutting with III were gel-eluted and ligated to the Hind III site of the vector prepared in step 5 to finally construct the lactic acid bacterium insertion vector pLT7I. The schematic diagram of the said vector pLT7I is shown in FIG.

Step 7: Lactobacillus Planta Room  Confirmation of insertion of T7 RNA polymerase gene into genome

After extracting genomic DNA of the transgenic lactic acid bacteria, PCR amplification was performed using the primer T7-N1 / T7-C1 used in the first step. As a result, it was confirmed that the 2.7 kb T7 RNA polymerase gene was inserted into the genome of the transgenic lactic acid bacteria. The results are shown in FIG. In addition, it was confirmed that the T7 RNA polymerase gene was inserted by Southern blotting. The results are shown in FIG.

Example 2 Construction of Recombinant Lactic Acid Bacteria Expression Vector Equipped with T7 RNA Polymerase Promoter and S Layer Signal Sequence

First step: Lactobacillus brevis Cloning the S-layer signal sequence from genomic DNA of

In order to clone the S layer signal sequence region known as a gene sequence that effectively secretes foreign proteins in lactic acid bacteria, the following primers were prepared and PCR amplification was performed to obtain three fragments. The results are shown in FIG.

PS (1-2): TT GAATTC ACCCAGCCAGTACCAGAAGC

                   EcoR I

PS (1-3): TT GGATCC AACGACAATCAGAGCGTAA

                   BamH I

PS (1-4): TT GGATCC TAACTTCGGTTATACTATTCTTG

                   BamH I

PS2-1: CGTA TCTAGA AGCTGAAGCAGTCGTTG

                   Xba I

Step 2: ligation of the fragment of step 1 to the front of the gene from which the signal sequence and promoter to be expressed are removed

5 'of the desired gene was inserted into the Xba I site and 3' into the Xho I site, and PCR amplification was carried out to obtain the product. Thereafter, the product was cut into Xba I and Xho I, ligated with the fragment of the first step, and then PCR amplification was performed again to obtain a new fragment ligated with the signal sequence. This fragment was inserted into the pGEM-T vector, transformed, and the inserted colonies were selected. Thereafter, the plasmid was extracted, cut with EcoR I / Xho I or BamH I / Xho I, and gel eluted to obtain the fragment.

Step 3: ligation of the T7 RNA polymerase promoter

The pET-21 vector with the T7 RNA polymerase promoter was cut with EcoR I / Xho I or BamH I / Xho I and gel eluted to ligate the fragment of the second step. After confirming the inserted colonies, primers ligated with the T7 RNA polymerase promoter, that is, primers based on the T7 RNA polymerase promoter site present in the pET21 vector, were prepared as follows and PCR products were obtained using the same. .

LPEN-2: TT GAATTC TAGGCGCCAGCAACCGCACC

                  EcoR I

LPEC-2: TT GTCGAC TTAATGCGCCGCTACAGGG

                  Sal I

Fourth step: ligation of the fragment of the third step into the lactic acid bacteria vector

E. coli and the shuttle vector of the lactic acid bacteria (shuttle vector) pNZ123 was cut into EcoR I and Sal I, and then ligated with the EcoR I / Sal I fragment of the third step to transform E. coli JM 101. Thereafter, insert-inserted colonies were selected, and plasmids were extracted from the transformed E. coli to transform the lactic acid bacteria into which the T7 RNA polymerase gene was inserted. The transformed lactic acid bacteria were then deposited to the Korean spawn association on February 20, 2002. (Accession No .: KCCM-10355) Recombinant plasmid vector equipped with T7 RNA polymerase promoter and S layer signal sequence [p123 ( 1-4) Endo] is shown in FIG.

Step 5: confirming the foreign protein expression of the transformed lactic acid bacteria

Since the cellulase gene was used as the reporter gene, the Congo red test was conducted to confirm whether the transgenic lactic acid bacteria (KCCM-10355) expressed cellulase. Cellulase activity of the present invention transformed lactic acid bacteria by Congo red test is shown in FIG. On the other hand, the overall expression schematic of the transformed lactic acid bacteria of the present invention is shown in FIG.

As described above, the present invention inserts a T7 RNA polymerase gene into the Lactobacillus plantarum genome, and is equipped with a T7 RNA polymerase promoter and an S-layer signal sequence capable of recognizing the T7 RNA polymerase. After constructing the plasmid vector, a final transformed lactic acid bacterium was transformed by transforming the plasmid vector into the transgenic lactic acid bacterium into which the T7 RNA polymerase gene was inserted. It is a very useful invention for the biotechnology industry because it can produce.

Claims (5)

delete delete delete A cleavage map prepared by inserting a T7 RNA polymerase gene into a pGEM-T vector and a lac promoter in front of the T7 RNA polymerase gene and inserting the lactic acid bacterium insertion vector pLT7I shown in FIG. , A cleavage map prepared by ligation of the S-layer signal sequence to the pGEM-T vector and insertion of the T7 RNA polymerase promoter, and the recombinant plasmid vector p123 (1-4) Endo shown in FIG. Transformed lactic acid bacteria (KCCM-10355) prepared by transformation. A cleavage map prepared by inserting a T7 RNA polymerase gene into a pGEM-T vector and a lac promoter in front of the T7 RNA polymerase gene and inserting the lactic acid bacterium insertion vector pLT7I shown in FIG. , A cleavage map prepared by ligation of the S-layer signal sequence to the pGEM-T vector and insertion of the T7 RNA polymerase promoter, and the recombinant plasmid vector p123 (1-4) Endo shown in FIG. Method for producing a transgenic lactic acid bacteria (KCCM-10355) characterized in that the transformation.

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