JP2009125008A - Method for producing hybrid polynucleotide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing various hybrid genes with decreased number of steps in contrast with conventional gene mutagenesis method necessitating complicated procedures. <P>SOLUTION: In a method for producing a hybrid polynucleotide by an in vivo homologous recombination in a microbial cell, a linear gene is formed by connecting only one terminal of the original polynucleotide to both ends of a linear vector in the same direction, and the gene is cyclized by homologous recombination to obtain a hybrid polynucleotide. In the second recombination or thereafter, only a hybrid polynucleotide having one terminal derived from the same polynucleotide is used to perform the recombination. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a hybrid polynucleotide, wherein a gene recombination method using homologous recombination efficiently introduces more gene mutations into a gene encoding an existing protein with fewer steps.

  Introducing a gene mutation into a gene encoding an existing protein to give it a new function has long been performed. In this method, DNA polymerase is called error-prone PCR (Plymerase Chain Reaction), which utilizes the property of reading a gene at a certain rate and mistakenly, and cells are treated with mutagenic substances or ultraviolet rays. There are ways to mutate genes.

  Attempts have also been made to improve proteins by replacing part of the amino acid sequence of existing proteins with other sequences. Among them, a method for producing a hybrid between two or more proteins belonging to the same protein family derived from a common ancestor molecule has been recently developed. The method involves fragmenting multiple genes (parent genes) that encode the protein of interest, and recombining the gene fragments in vitro by PCR to generate a large number of hybrid genes in which multiple parent genes are randomly linked. There is a method called Sexal PCR or family shuffling that can be prepared (for example, see Patent Document 1), and a method of forming a hybrid gene more efficiently by performing fragmentation of a gene with a restriction enzyme (for example, Patent Document 2).

  On the other hand, using homologous recombination in microorganisms such as Escherichia coli, a hybrid gene is created by replacing part of the gene sequence of an existing protein with another sequence in vivo, and the existing protein has a new function. It has been tried. There are many methods for producing a hybrid gene using homologous recombination by RecA protein in Escherichia coli. In this method, a plasmid in which two types of homologous genes are cloned in the same direction on a vector is prepared, a linear gene is obtained by cleaving the two genes with a restriction enzyme, and then the linear gene Is introduced into Escherichia coli, and the gene is circularized by homologous recombination between homologous genes using RecA protein to obtain a hybrid gene. In this method, a hybrid gene can be prepared from genes having relatively low homology. Also, a method for preparing a linear DNA by preparing a linear vector in advance and ligating only one end of the gene fragment to the circular vector containing the gene fragment instead of cleaving with a restriction enzyme. (For example, refer to Patent Document 3). In this method, it is not necessary to clone a gene every time homologous recombination occurs, and it is possible to produce a hybrid gene of a plurality of combinations in one reaction.

Japanese National Patent Publication No. 10-50561 JP 2000-245473 A JP 2006-42737 A

  These various gene mutation introduction methods and hybrid gene production methods are all effective methods, but there are some problems to be improved.

  In other words, error-prone PCR, cell processing with mutagenic substances and ultraviolet rays occur due to gene mutation or randomization, so that the functional structure of the target protein is destroyed or cells are killed. There is. In existing family shuffling in vitro, there is a complicated process in which an unspecified site or a specific site of a gene must be cleaved by some method to fragment the gene. Further, since gene fragments are recombined using PCR in a test tube, a relatively high homology of 80% or more is required even in the case of fragmentation with a restriction enzyme. Therefore, although it is effective for producing a hybrid of gene sequences having high homology, it may not be suitable for producing a hybrid gene between genes having low homology. On the other hand, in the method using homologous recombination in vivo, only one homologous recombination occurs in one reaction, and there are fewer types of hybrid genes obtained compared to the family shuffling method, When performing the second and third homologous recombination in order to increase the number of types, there is a problem that the gene must be cloned each time.

  In addition, a linear vector is prepared in advance, and only one end of each of two types of gene fragments (assuming the first polynucleotide and the second polynucleotide, respectively) are linked to form a linear vector. In the method for preparing the DNA, the polynucleotide obtained by the two recombination is derived from the first polynucleotide at the 5 ′ end portion, from the second polynucleotide at the central portion, and from the 3 ′ end portion. If only the hybrid derived from the first polynucleotide is to be introduced and further mutations are to be introduced, further recombination must be performed. In addition, when performing the second and subsequent recombination, a chimeric gene in which the 5 ′ end portion of the linear gene is derived from the first polynucleotide and a chimeric gene in which the 3 ′ end portion is derived from the first polynucleotide Both must be prepared and are cumbersome.

  An object of the present invention is to provide a method for producing a hybrid polynucleotide as a means for easily producing more diverse hybrid genes with fewer steps based on a method using in vivo homologous recombination in a microbial cell. It is to provide.

  In the conventional homologous recombination method in microorganisms, two types of homologous genes are cloned into dandem, and then cleaved with a restriction enzyme to obtain linear DNA. A chimeric gene in which the 5′-end portion is derived from the first polynucleotide after being circularized by homologous recombination, and the 3′-end portion is derived from the first polynucleotide. Was used for the second and subsequent recombination.

  The present inventors have found a method for easily preparing various hybrid genes by using only a chimeric gene whose 5 ′ terminal portion is derived from the first polynucleotide in the second and subsequent recombination. It was.

  That is, the present invention provides a chimeric gene in which the 5 ′ end portion is derived from the first polynucleotide and the 3 ′ end portion is derived from the first polynucleotide in the second and subsequent recombination as in the conventional homologous recombination method. It is not necessary to prepare both of the chimeric genes to be prepared, and a variety of hybrid genes can be produced.

  Therefore, the present invention is a polynucleotide hybrid production method for easily producing more diverse hybrid genes with fewer steps, based on a polynucleotide hybrid production method using in vivo homologous recombination in microbial cells. is there. Hereinafter, the present invention will be described in detail.

  Each of the first polynucleotide and the second polynucleotide in the present invention may be one kind or two kinds or more.

  Although the arbitrary vector in this invention is not specifically limited, Usually, what contains marker genes, such as an antibiotic resistance gene, is used.

  The microorganism having homologous recombination ability in the present invention is not particularly limited, but usually an Escherichia coli strain having a recA gene is used, particularly preferably a recBCsbcA mutant E. coli.

  The restriction enzymes that cleave both ends of the first polynucleotide and the second polynucleotide used in the method described in claim 1 of the present invention are preferably different restriction enzymes. The restriction enzyme that cuts the 5 ′ end or the restriction enzyme that cuts the 3 ′ end may be the same restriction enzyme. Moreover, the restriction enzymes used in the method described in claim 2 of the present invention are different restriction enzymes. When each step in the method of the present invention is repeated twice or more, the restriction enzyme used is usually the same as that used for the first time, but different restriction enzymes may be used.

  The outline of the method according to claim 1 of the present invention will be described with reference to FIGS. The present invention will be described with reference to an example in which a first polynucleotide and a second polynucleotide having a partial sequence homologous thereto are cloned into a multiple cloning site of the vector. Applicable to all gene fragments with Different restriction enzymes are described as restriction enzymes A, B, C, and D. Restriction enzyme A is a restriction enzyme that cleaves the gene at the 5 ′ end of the first polynucleotide or upstream thereof. Restriction enzyme B is a restriction enzyme that cleaves the gene at the 3 ′ end of the first polynucleotide or downstream thereof. Enzyme, restriction enzyme C is a restriction enzyme that cleaves the gene at the 5 ′ end side or upstream of the second polynucleotide, and restriction enzyme D is a gene at the 3 ′ end side or downstream of the second polynucleotide Is a restriction enzyme that cleaves.

  A vector cloned from the first polynucleotide is treated with restriction enzyme B, followed by dephosphorylation of the cleavage site, followed by treatment with restriction enzyme A to cleave the polynucleotide from the vector. Fragments are prepared (shown as the first polynucleotide in FIGS. 1, 1-1). Similarly, a vector obtained by cloning the second polynucleotide is treated with restriction enzyme C and then dephosphorylated by treating the cleavage site with alkaline phosphatase, and then treated with restriction enzyme D, and the polynucleotide is vectorized. A second polynucleotide fragment is prepared by detaching from (shown as the second polynucleotide in FIGS. 1, 1-1). The alphabet at the end of each polynucleotide indicates a cleaved restriction enzyme site. An asterisk (*) indicates a dephosphorylation site. A vector is prepared by cleaving with enzymes A and D and dephosphorylated by treating with alkaline phosphatase (shown as vectors in FIGS. 1 and 1-2). When the first polynucleotide and the second polynucleotide are mixed with the vector and ligated, the B * and C * portions cannot be linked, so that a linear molecule is formed (shown as FIGS. 1 and 1-3). After the ligation reaction, only the target linearly linked molecules are collected by agarose gel electrophoresis or the like, introduced into a microorganism having homologous recombination ability, and applied to a medium containing an antibiotic corresponding to the vector. Homologous recombination occurs at the homologous part of the two polynucleotides, and only E. coli having a circular molecule grows. Plasmids are collectively collected from colonies grown in a medium containing antibiotics and purified in bulk to form a single recombinant hybrid polynucleotide library 1-1 (shown as FIGS. 1, 1-4). Plasmid extraction may be performed by a general extraction method from E. coli, and may be performed using a commercially available plasmid extraction kit (QIAprep, manufactured by Qiagen). The polynucleotide library 1-1 is composed of a hybrid in which the 5 ′ end portion is derived from the first polynucleotide and the 3 ′ end portion is derived from the second polynucleotide (shown as FIGS. 1 and 1-4). .

  Subsequently, a second recombination is performed on the hybrid polynucleotide library prepared by the first recombination. The method is the same as the first method.

  The polynucleotide library 1-1 is treated with restriction enzyme B and then dephosphorylated by treating the cleavage site with alkaline phosphatase, and then treated with restriction enzyme A to cleave the polynucleotide from the vector, A polynucleotide fragment is prepared (shown as the first polynucleotide in FIGS. 2, 2-1). Similarly, the polynucleotide library 1-1 is treated with restriction enzyme C and then dephosphorylated by treating the cleavage site with alkaline phosphatase, then treated with restriction enzyme D to cleave the polynucleotide from the vector, A second polynucleotide fragment is prepared (shown as the second polynucleotide in FIGS. 2, 2-1). Prepare a vector that has been dephosphorylated by digestion with enzymes A and D and treatment with alkaline phosphatase (shown as vectors in FIGS. 2 and 2-2). When the first polynucleotide and the second polynucleotide are mixed with the vector and ligated, the B * and C * portions cannot be linked, so that a linear molecule is formed (shown as FIGS. 2 and 2-3). After the ligation reaction, only the target linearly linked molecules are collected by agarose gel electrophoresis or the like, introduced into a microorganism having homologous recombination ability, and applied to a medium containing an antibiotic corresponding to the vector. Homologous recombination occurs at the homologous part of the two polynucleotides, and only E. coli having a circular molecule grows. Plasmids are collectively collected from colonies grown on antibiotic-containing media and purified in bulk to yield a twice-recombinant hybrid polynucleotide library 1-2 (shown as FIGS. 2, 2-4). The polynucleotide portion of the polynucleotide library 1-2 has a 5 ′ terminal portion derived from the first polynucleotide, followed by a portion derived from the second polynucleotide, followed by a portion derived from the first polynucleotide. A clone consisting of a hybrid whose 3 ′ end portion is derived from a second polynucleotide, and a hybrid whose 5 ′ end portion is derived from a first polynucleotide and whose 3 ′ end portion is derived from a second polynucleotide It consists of a clone. (Shown as FIGS. 2, 2-4).

  For the hybrid polynucleotide library prepared by the second recombination, perform the third recombination, the fourth recombination for the hybrid polynucleotide library prepared by the third recombination, and then the recombination sequentially. It can be carried out. The method is the same as the first recombination. This makes it possible to easily produce more diverse hybrid genes with fewer steps.

  Moreover, the same effect can be expected even if the first polynucleotide and the second polynucleotide are replaced and recombination is performed by the same operation.

  The outline of the method according to claim 2 of the present invention will be described with reference to FIGS. The present invention will be described by taking as an example the cloning of a first polynucleotide and a second polynucleotide having a partial sequence homologous thereto into a vector multiple cloning site. However, the present invention covers all homologous gene fragments. Applicable to. Different restriction enzymes will be described as restriction enzymes E, H, P, and F. Restriction enzyme E is a restriction enzyme that cleaves the gene at the 5 ′ end of the first polynucleotide or upstream thereof, restriction enzyme H is a gene that cleaves the gene at the 3 ′ end of the first polynucleotide or downstream thereof. Restriction enzyme P, restriction enzyme P is a restriction enzyme that cleaves the gene at the 3 ′ end of the second polynucleotide or downstream thereof and cleaves a portion closer to the second polynucleotide than restriction enzyme H F is a restriction enzyme that cleaves the gene at the 5 ′ end of the second polynucleotide or upstream thereof and cleaves the portion closer to the second polynucleotide than the restriction enzyme E. The first polynucleotide fragment is prepared by cleaving the vector cloned from the first polynucleotide with restriction enzymes E and H, cleaving the polynucleotide from the vector, and dephosphorylating the phosphate groups at both ends. (Shown as the first polynucleotide in FIGS. 3, 3-1). A vector in which a second polynucleotide having a sequence homologous to the first polynucleotide is cloned, is dephosphorylated by cleaving with a restriction enzyme P and then treating the phosphate group at the cleavage site with alkaline phosphatase; Next, a vector gene containing the second polynucleotide fragment is prepared by treating with restriction enzyme H (shown as the second polynucleotide in FIGS. 3 and 3-1). The alphabet at the end of each polynucleotide indicates a cleaved restriction enzyme site. An asterisk (*) indicates a dephosphorylation site. When a vector containing the first polynucleotide and the second polynucleotide is mixed and ligated, binding occurs only at the restriction enzyme H cleavage site to form a linear molecule (shown as FIGS. 3 and 3-2). After the ligation reaction, only the target linearly linked molecules are collected by agarose gel electrophoresis or the like, introduced into a microorganism having homologous recombination ability, and applied to a medium containing an antibiotic corresponding to the vector. Homologous recombination occurs at the homologous part of the two polynucleotides, and only E. coli having a circular molecule grows. Plasmids are collectively collected from colonies grown in a medium containing antibiotics and purified in bulk to give a single recombinant hybrid polynucleotide library 3-1 (shown as FIGS. 3 and 3-3). Polynucleotide library 3-1 consists of hybrids whose 5 'end portion is derived from the second polynucleotide and whose 3' end portion is derived from the first polynucleotide (shown as FIGS. 3, 3-3).

  Subsequently, a second recombination is performed on the hybrid polynucleotide library prepared by the first recombination. The method is the same as the first method.

  A first polynucleotide fragment is prepared by cleaving the polynucleotide library 3-1 with restriction enzymes E and H, cleaving the polynucleotide from the vector, and dephosphorylating the phosphate groups at both ends (FIG. 4, 4-1 shown as the first polynucleotide). On the other hand, after cleaving the polynucleotide library 3-1 with the restriction enzyme P, the phosphate group at the cleavage site is treated with alkaline phosphatase, and then dephosphorylated, and then treated with the restriction enzyme H to produce a second enzyme. A vector gene containing the polynucleotide fragment is prepared (shown as the second polynucleotide in FIGS. 4 and 4-1). When a vector containing the first polynucleotide and the second polynucleotide is mixed and ligated, binding occurs only at the cleavage site by the restriction enzyme H to form a linear molecule (shown as FIGS. 4 and 4-2). After the ligation reaction, only the target linearly linked molecules are collected by agarose gel electrophoresis or the like, introduced into a microorganism having homologous recombination ability, and applied to a medium containing an antibiotic corresponding to the vector. Homologous recombination occurs at the homologous part of the two polynucleotides, and only E. coli having a circular molecule grows. Plasmids are collectively collected from colonies grown in a medium containing antibiotics and purified in bulk to produce a single recombinant hybrid polynucleotide library 3-2 (shown as FIGS. 4 and 4-3).

  The polynucleotide portion of the polynucleotide library 3-2 has a 5 ′ terminal portion derived from the first polynucleotide, followed by a portion derived from the second polynucleotide, followed by a portion derived from the first polynucleotide. A clone consisting of a hybrid whose 3 ′ end portion is derived from a second polynucleotide, and a hybrid whose 5 ′ end portion is derived from a first polynucleotide and whose 3 ′ end portion is derived from a second polynucleotide It consists of a clone. (Shown as FIGS. 4, 4-3).

  For the hybrid polynucleotide library prepared by the second recombination, perform the third recombination, the fourth recombination for the hybrid polynucleotide library prepared by the third recombination, and then the recombination sequentially. It can be carried out. The method is carried out by using a polynucleotide library in which the 5 ′ end of the polynucleotide to be recombined is derived from either the first polynucleotide or the second polynucleotide as in the second recombination. Good. This makes it possible to easily produce more diverse hybrid genes with fewer steps.

  In addition, the vector in which the second polynucleotide is cloned is cleaved with a restriction enzyme F that cleaves at the 5 ′ end of the second polynucleotide, and the cleavage site is treated with alkaline phosphatase to dephosphorylate, Using the vector gene containing the second polynucleotide fragment obtained by cleaving the 3 ′ end of the vector with restriction enzyme E, ligation and homologous recombination with the first polynucleotide shown in FIG. Similar effects can be expected.

  A hybrid polynucleotide library similar to the method of claim 2 of the present invention can also be prepared by a method obtained by changing a part of the method of claim 2 of the present invention described above. Hereinafter, a method obtained by changing a part of the method according to claim 2 of the present invention will be described with reference to FIG. The present invention will be described by taking as an example the cloning of a first polynucleotide and a second polynucleotide having a partial sequence homologous thereto into a vector multiple cloning site. However, the present invention covers all homologous gene fragments. Applicable to. Different restriction enzymes will be described as restriction enzymes E, H, P, and F. Restriction enzyme E is a restriction enzyme that cleaves the gene at the 5 ′ end of the first polynucleotide or upstream thereof, restriction enzyme H is a gene that cleaves the gene at the 3 ′ end of the first polynucleotide or downstream thereof. Restriction enzyme P, restriction enzyme P is a restriction enzyme that cleaves the gene at the 3 ′ end of the second polynucleotide or downstream thereof and cleaves a portion closer to the second polynucleotide than restriction enzyme H F is a restriction enzyme that cleaves the gene at the 5 ′ end of the second polynucleotide or upstream thereof and cleaves the portion closer to the second polynucleotide than the restriction enzyme E.

  The vector obtained by cloning the first polynucleotide is cleaved with the restriction enzyme E, and the phosphate group at the cleavage site is treated with alkaline phosphatase to be dephosphorylated, and then further cleaved with the restriction enzyme H. From the vector (shown as the first polynucleotide in FIGS. 5 and 5-1). The second polynucleotide is obtained by cleaving the vector cloned from the second polynucleotide having a sequence homologous to the first polynucleotide with restriction enzymes P and H and then dephosphorylating the phosphate group at the cleavage site. A vector gene containing the fragment is prepared (shown as the second polynucleotide in FIGS. 5 and 5-1). The alphabet at the end of each polynucleotide indicates a cleaved restriction enzyme site. An asterisk (*) indicates a dephosphorylation site. When a vector containing the first polynucleotide and the second polynucleotide is mixed and ligated, binding occurs only at the site cleaved by the restriction enzyme H to form a linear molecule (shown as FIGS. 5 and 5-2). After the ligation reaction, only the target linearly linked molecules are collected by agarose gel electrophoresis or the like, introduced into a microorganism having homologous recombination ability, and applied to a medium containing an antibiotic corresponding to the vector. Homologous recombination occurs at the homologous part of the two polynucleotides, and only E. coli having a circular molecule grows. Plasmids are collectively collected from colonies grown in a medium containing antibiotics and purified in bulk to yield a single recombinant hybrid polynucleotide library 4-1 (shown as FIGS. 5 and 5-3). Polynucleotide library 4-1 consists of a hybrid whose 5 'end portion is derived from the second polynucleotide and whose 3' end portion is derived from the first polynucleotide (shown as FIGS. 5, 5-3).

  Subsequently, a second recombination is performed on the hybrid polynucleotide library prepared by the first recombination. The method is the same as the first method. In the second and subsequent recombination, a polynucleotide library in which the 5 ′ end of the polynucleotide to be recombined is derived from either the first polynucleotide or the second polynucleotide is used as described above. By performing the above, a hybrid polynucleotide library similar to the method of claim 2 of the present invention can be obtained. This makes it possible to easily produce more diverse hybrid genes with fewer steps. In addition, the vector in which the second polynucleotide is cloned is cut with a restriction enzyme F that cuts at the 5 ′ end of the second polynucleotide or a gene upstream thereof, and the cleavage site is treated with alkaline phosphatase. After dephosphorylation, ligation and homologous recombination with the first polynucleotide using a vector gene containing the second polynucleotide fragment obtained by cleaving the 3 ′ end of the vector with restriction enzyme E is the same. The effect can be expected.

  According to the present invention, more diverse hybrid genes can be easily produced with fewer steps by utilizing homologous recombination in E. coli without fragmenting the genes. The present invention has functions of existing proteins, such as the creation of antibodies with excellent affinity and specificity, the creation of enzymes with excellent heat resistance and enzymes with altered substrate specificity, and completely new functions. It is effective in creating new proteins.

  Examples are shown below to describe the invention in more detail, but the invention is not limited to these Examples.

Example 1 Preparation of Linear Antibody Gene for First Recombination Two homologous antibody genes E06 and E30 against the same antigen were cloned into a multiple cloning site of vector pFCAH10 to obtain pE06 and pE30, respectively. Plasmid pE06 from which the gene was cloned was cleaved with restriction enzyme XhoI and restriction enzyme NheI, purified and then dephosphorylated with shrim alkalin phosphatase (SAP) to obtain pE06 (X * / N *).

  The plasmid pE30 was cleaved with the restriction enzyme SfiI, purified and then dephosphorylated with SAP. Further, it was cleaved with the restriction enzyme NheI to obtain pE30 (S * / N). This was mixed with the above-mentioned pE06 (X * / N *), and a ligation reaction was performed using T4 ligase. After the reaction, the entire amount was subjected to agarose gel electrophoresis, and a 5.7 kb fragment was recovered from the gel and purified by phenol extraction and ethanol precipitation.

Example 2 Preparation of Single Recombinant Hybrid Gene Library The linear gene prepared in Example 1 was introduced into the recBCsbcA mutant JC8679 of E. coli by electroporation. Colonies were grown on a medium containing carbenicillin, colonies were randomly selected, plasmids were purified from each colony, and the base sequences of gene fragments on the plasmids were determined. As a result, a hybrid gene in which the 5 ′ end portion was derived from pE06 and the 3 ′ end portion was derived from pE30 was formed (6-1 in FIG. 6).

  Therefore, a plasmid was prepared from these transformed bacteria grown on a medium containing carbenicillin, and it was designated as a single recombinant hybrid gene library pE06 / 30-1 and a clone No. such as E06 / 30-1-14. Was granted.

Example 3 Preparation of Linear Antibody Gene for Second Recombination Hybrid gene library pE06 / 30-1 was cleaved with XhoI and NheI, purified and dephosphorylated with shrim alkalin phosphatase (SAP), pE06 / 30-1 (X * / N *) was obtained. The hybrid gene library pE06 / 30-1 was cleaved with SfiI, purified and then dephosphorylated with SAP. Cleavage with NheI gave pE06 / 30-1 (S * / N). This was mixed with the above pE06 / 30-1 (X * / N *), and a ligation reaction was performed using T4 ligase. After the reaction, the entire amount was subjected to agarose gel electrophoresis, and a 5.7 kb fragment was recovered from the gel and purified by phenol extraction and ethanol precipitation.

Example 4 Preparation of Double Recombinant Hybrid Gene Library The linear gene prepared in Example 3 was introduced into the recBCsbcA mutant JC8679 of E. coli by electroporation. Colonies were grown on a medium containing carbenicillin, colonies were randomly selected, plasmids were purified from each colony, and the base sequences of gene fragments on the plasmids were determined.

  As a result, a gene having a 5 ′ end portion derived from pE06, followed by a portion derived from pE30, followed by a portion derived from pE06, a 3 ′ end portion comprising a hybrid derived from pE30, and a 5 ′ end portion A hybrid gene derived from pE06 and having a 3'-terminal portion derived from pE30 was formed (6-2 in FIG. 6).

  Therefore, a plasmid was prepared from these transformed bacteria grown in a medium containing carbenicillin, and it was designated as a twice-recombinant hybrid gene library pE06 / 30-2, with a clone number such as E06 / 30-2-4. Was granted.

Example 5 Production of Third Recombination Hybrid Gene Library Similarly, the third homologous recombination was performed in the same manner as the first and second homologous recombination using the second recombination hybrid gene library pEpE06 / 30-2. It was. As a result, the 5 ′ end portion different from the second one is derived from pE06, followed by a portion derived from pE30, followed by a portion derived from pE06, and a 3 ′ end portion comprising a hybrid derived from pE30. And a hybrid gene in which the 5 ′ end portion was derived from pE06 and the 3 ′ end portion was derived from pE30 (6-3 in FIG. 6).

  Therefore, a plasmid was prepared from these transformed bacteria grown in a medium containing carbenicillin, and the three-fold recombinant hybrid gene library pE06 / 30-3 was used, and the branch number such as E06 / 30-3-33 was used as the clone No. Was granted.

It is a figure which shows the outline | summary of the method of Claim 1, and is a figure which shows the method regarding the recombination of the 1st time. In FIG. Restriction enzyme, B is a restriction enzyme that cleaves the gene at the 3 ′ end of the first polynucleotide or downstream thereof, and C is a restriction enzyme that cleaves the gene at the 5 ′ end of the second polynucleotide or upstream thereof. Enzyme D is a restriction enzyme that cleaves the gene at the 3 ′ end of the second polynucleotide or downstream thereof, an asterisk (*) indicates a dephosphorylation site, and a white square indicates the first poly-nucleotide. Nucleotides, black squares indicate the second polynucleotide. It is a figure which shows the outline | summary of the method of Claim 1, and is a figure which shows the method regarding the 2nd recombination, A, B, C, D, a star (*), a white square, a black square in FIG. These squares are the same as those in FIG. It is a figure which shows the outline | summary of the method of Claim 2, and is a figure which shows the method regarding 1st recombination, and E cuts the gene of the 5 'terminal side of a 1st polynucleotide, or its upstream from FIG. Restriction enzyme, H, which cleaves the gene at the 3 ′ end of the first polynucleotide or downstream thereof, P, cleaves the gene at the 3 ′ end of the second polynucleotide, or downstream thereof And a restriction enzyme that cleaves the portion closer to the second polynucleotide than the restriction enzyme H, the asterisk (*) indicates the dephosphorylation site, the open square indicates the first polynucleotide, Squares indicate the second polynucleotide. It is a figure which shows the outline | summary of the method of Claim 2, and is a figure which shows the method regarding the 2nd recombination, and is E, H, P, an asterisk (*), a white square, a black square in FIG. Is the same as FIG. The outline of the method which changed a part of method of Claim 2 of this invention is shown, It is a figure which shows the method regarding recombination of the 1st time, E, H, P, an asterisk (*), an open square, The black squares are the same as in FIG. FIG. 4 shows the structure of the hybrid gene of pE06 and pE30 obtained in Examples 1 to 5, each square indicates a gene before or after recombination, and the open square indicates a polynucleotide derived from the antibody gene E06, which is black. Open squares indicate the polynucleotide derived from the antibody gene E30, and the horizontal axis indicates the length of the gene.

Claims (2)

A method for producing a hybrid polynucleotide, comprising the following steps (1) to (8):
(1) producing a first polynucleotide in which both ends are cleaved with different restriction enzymes and the 3 ′ end is dephosphorylated;
(2) producing a second polynucleotide having a sequence homologous to the first polynucleotide, wherein both ends are cleaved with different restriction enzymes and the 5 ′ end is dephosphorylated;
(3) contacting any vector with a restriction enzyme cleaved at the 5 ′ end of the first polynucleotide and a restriction enzyme cleaved at the 3 ′ end of the second polynucleotide to cleave the vector;
(4) Step of dephosphorylating both ends of the linear vector prepared in step (3) (5) Linear vector dephosphorylated at both ends prepared in step (4) and step (1 ) And the second polynucleotide prepared in the step (2) are brought into contact with each other, and the first polynucleotide is put on the 3 ′ end of the linear vector through the same restriction enzyme. Linking a second polynucleotide to the 5 ′ end of the linear vector to produce a linear molecule,
(6) The linear molecule produced in step (5) is introduced into a microorganism having homologous recombination ability, and homologous sequences existing on the first and second polynucleotides in the linear molecule are introduced. A step of selecting a microorganism in which the linear molecule has been circulated by causing homologous recombination at a portion thereof,
(7) A step of collecting a vector containing a hybrid polynucleotide from the microorganism selected in step (6),
(8) The polynucleotides collected in step (7) are used as the first polynucleotide and the second polynucleotide, and steps (1) to (7) are repeated twice or more. In both the first polynucleotide and the second polynucleotide of the present invention, the 5 ′ end is used to recombine only the polynucleotide derived from either the first polynucleotide or the second polynucleotide used in the previous recombination. Step to use. A method for producing a hybrid polynucleotide, comprising the following steps (1) to (6):
(1) both ends cleaved with different restriction enzymes (a) both the 5 ′ end and 3 ′ end are dephosphorylated, or (b) only the 5 ′ end is dephosphorylated, or (C) producing a first polynucleotide in which only the 3 ′ end is dephosphorylated,
(2) (a) When both the 5 ′ end and 3 ′ end of the first polynucleotide are dephosphorylated, the second polynucleotide having a sequence homologous to the first polynucleotide is changed to 3 Included on the end side, 5 ′ end is a cleavage end by a restriction enzyme cleaved from the 3 ′ end of the first polynucleotide, 3 ′ end is a cleavage end by another restriction enzyme, and 3 ′ end is dephosphorylated A linear vector that has been oxidized, or a second polynucleotide having a sequence homologous to the first polynucleotide at the 5 ′ end, and a cleavage end by a restriction enzyme having a 5 ′ end, and the 3 ′ end A linear vector having a restriction endonuclease cleaved at the 5 ′ end of the first polynucleotide and dephosphorylated at the 5 ′ end is prepared; (b) 5 ′ of the first polynucleotide When only the end is dephosphorylated Contains the second polynucleotide at the 3 ′ end, the 5 ′ end is the restriction end cut by the restriction enzyme that cuts the 3 ′ end of the first polynucleotide, and the 3 ′ end is the end cut by the other restriction enzyme. A linear vector in which both ends are dephosphorylated, and (c) when only the 3 ′ end of the first polynucleotide is dephosphorylated, The 'end side contains 5' end, the 3 'end is a restriction endonuclease cleaved by the restriction endonuclease that cleaves the 5' end of the first polynucleotide, and both ends are dephosphorylated. Producing a linear vector,
(3) a step of preparing a linear molecule by mixing the linear vector prepared in step (2) and the first polynucleotide and ligating each through a cleavage end with the same restriction enzyme,
(4) The linear molecule produced in step (3) is introduced into a microorganism having homologous recombination ability, and the homologous sequences present on the first and second polynucleotides in the linear molecule are A step of selecting a microorganism in which the linear molecule has been circulated by causing homologous recombination at a portion possessed, and a step of collecting a vector containing a hybrid polynucleotide from the microorganism selected in step (4).
(6) The polynucleotide collected in step (5) is used as the first polynucleotide and the second polynucleotide, and the steps (1) to (5) are repeated twice or more. In both the first polynucleotide and the second polynucleotide of the present invention, the 5 ′ end is used to recombine only the polynucleotide derived from either the first polynucleotide or the second polynucleotide used in the previous recombination. Step to use.

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