CN1165622C - A zero-background high-throughput directional expression cloning method - Google Patents

Abstract

The present invention proposes a zero-background, high-throughput, directional cloning and expression method, which is based on a specific restriction endonuclease double-cutting expression vector, using _T4 DNA polymerase to digest the PCR product amplified by specially designed primers products, and then directional cloning of PCR products into expression vectors. The specific restriction endonuclease can be any two (same or different) of Cpo I, Not I, Ear I, Sap I, Sty I. The invention is compatible with all PCR-based library construction methods currently reported, can achieve zero background, high-throughput directional cloning, and has a short experimental period and low cost, and is suitable for bacteria, fungi, animals, plants, etc. as hosts high-throughput cloning and expression. Is a very economical cloning method.

Description

A zero-background high-throughput directional expression cloning method

Technical field

The invention relates to biological gene cloning and expression technology, in particular to a zero-background high-throughput directional expression cloning method. It is especially suitable for directly constructing an expression library using PCR products.

Background information

Genome sequencing provides a large number of new genes, and there is an urgent need to develop new technologies to study the functions of genes and proteins on a large scale. Therefore, many companies are committed to developing high-throughput cloning and expression technologies.

At present, several companies have launched high-throughput cloning and expression systems, such as Invitrogen's Echo ^TM system, Gibco/Life Technologies' Gateway ^TM system, and Novagen pTriEx-1 cloning system. The technical routes adopted by these systems are similar. That is, after cloning the PCR product, use the Cre enzyme to mediate the homologous recombination of the LoxP site to construct an expression vector, without the need for restriction endonuclease-mediated subcloning, and can construct a variety of expression vectors with high throughput. This improves the experimental efficiency and reduces the experimental cost to a certain extent. However, these methods have shortcomings, mainly in the following three aspects:

1. The experiment period is long, the cloning efficiency is low, and zero background and directional cloning cannot be performed. The first step of gene cloning in the above high-throughput cloning and expression system adopts the method of conventional PCR amplification and ligase-mediated ligation. In order to improve the ligation efficiency, a lethal gene is often introduced into the vector to eliminate the background of the self-ligation of the vector. Cloning PCR products with T vectors is a very effective method, but using T vectors, high-fidelity DNA polymerases cannot be used to amplify the target gene, because only DNA polymerases lacking 3'---5' corrective enzyme activity can be A base A is added to the 3' end of the PCR amplification product, which is complementary to the protruding T at the 3' end of the T vector, which results in increased PCR incorporation errors. In addition, the target gene cannot be directional cloned into an expression vector, so these methods require complex screening to obtain the correct clone. Invitrogen's cloning method based on five-minute quick ligation mediated by topoisomerase I (Topoisomerase I) is considered to be the most advanced cloning method at present, but it does not solve the problems of cloning fidelity and directionality. After the target gene amplified by PCR is cloned into the topoisomerase carrier, it is necessary to find out the clones with the gene inserted in the correct direction by PCR, and then randomly select several clones from these correct clones for sequencing analysis to determine whether the cloned gene contains Mutations introduced by PCR; it is well known that DNA polymerases lacking 3'---5' proofreading enzyme activity have an error rate of 0.1% during each round of PCR amplification (while the retention rate of high-fidelity DNA polymerases (such as Pfu) Authenticity is more than 10 times that of DNA polymerase lacking 3'---5' correction enzyme activity), so sequencing analysis is inevitable, so that at least It takes three days. In addition, because the base sequence recognized by topoisomerase is CCCTT, and it catalyzes a reversible reaction, if the cloning fragment is large, the cloning efficiency will be greatly reduced. In 1999, when John AHeyman et al. used this method to clone and express 6035 open reading frames of brewer's yeast on a large scale, only 75% of the open reading frames were inserted into clones in the correct direction, and only 41% of the clones were successfully expressed in yeast, so The success rate of the whole experiment ended up being only 30%.

2. The method of constructing expression vectors by Cre enzyme-mediated homologous recombination of loxP sites can be widely used in constructing expression vectors with bacteria, fungi, animals, plants, etc. as hosts. It is a high-throughput method, but it also has With defects:

1. It is mainly suitable for fusion expression. The target gene is generally fused at the C-terminus of the loxP site, and the N-terminus of the expressed target protein will have more than ten amino acids, which must be different from the conformation and function of the natural protein.

2. The loxP site is an inverted repeat sequence that can form a stem-loop structure, which reduces translation efficiency and is difficult to adapt to non-fusion expression.

3. It is difficult to construct prokaryotic expression vectors and eukaryotic expression non-fusion vectors in parallel. The introduction of loxP sites upstream of the start codon will inevitably introduce more than 30 additional nucleotides, which cannot meet the distance requirements of prokaryotic SD sequences and eukaryotic sequences at the same time. Consistency requirements for Kozak sequences.

Third, the experimental cost is high, which is not economical for large-scale research. The topoisomerase vector used for gene cloning is expensive ($19/reaction), and the Cre enzyme that mediates homologous recombination is also expensive ($10/reaction). PCR is used to identify the cloning direction and select the correct opening by sequencing analysis. Reading frames greatly increase research costs.

(See the publication "Genome Research, Vol. 9, 1999, pp. 383-392";

"Protein Expression and Purification, Vol. 22, 2001, pp. 159--164")

Contents of Invention

The purpose of the present invention is to provide a technology for biological gene expression cloning, which can truly achieve zero-background, high-throughput directional cloning, and can simultaneously construct prokaryotic expression vectors and eukaryotic expression vectors, which can not only fusion expression, but also fusion expression.

The present invention is achieved like this. Select an expression vector and target gene according to the target requirements of cloning. Two special restriction endonuclease cut site sequences and one lethal gene expression unit were introduced into the expression vector; 3--5 specific specific enzymes were introduced at the 5' end of a pair of PCR amplification primers of the target gene. In the presence of dATP (or dGTP or dCTP or dTTP), the sticky end of the amplified product after digestion with T ₄ DNA polymerase matches the sticky end obtained by double enzyme digestion of the vector, so that directional cloning can be performed in The vector was double-digested with two specific restriction enzymes.

Specifically, an expression vector and target gene are selected. On the expression vector, a polynucleotide linker is firstly designed, a Cpo I and Not I restriction site is introduced at both ends, and an expression unit of a lethal gene is connected in the middle, and the linker is introduced into the expression vector. The vector is digested with Cpo I and Not I to generate two cohesive ends:

3′ GC——————CGCCGG 5′

5′GTCCG—————GC 3′;

The 5' ends of a pair of PCR primers for amplifying the target gene are respectively introduced with 4-5 nucleotides such as 5'GACT3' and 5'GGCCT3', and the products obtained by PCR amplification are added with dATP and _T4 DNA polymerase, produces sticky ends like this:

5′GACT——————A 3′

3′ A—————TCCGG 5′;

The sticky end can just match with the sticky end produced by double enzyme digestion of the vector, so that the PCR product can be directional cloned into the vector.

The restriction endonucleases at both ends of the polynucleotide linker can be any two of Cpo I, Ear I, Not I, Sap I, Sty I or any one of Cpo I, Ear I, Sap I, Sty I. Any restriction endonuclease that can obtain no more than 3 (including 3) base types at the 5' protruding cohesive end after digestion can be used.

Compared with the prior art, the present invention has obvious advantages:

1. Can achieve zero background, high-throughput directional cloning. Firstly, the PCR primers and cloning vectors have been specially designed, and each pair of PCR primers can introduce 2-5 specific nucleotides at its 5′ end, and the obtained PCR products are treated with _T4 DNA polymerase, There will be sticky ends that do not match each other, and a linker is introduced into the vector, and the sticky ends produced after digestion with a special restriction endonuclease can just match the sticky ends of the processed PCR products, enabling directional cloning of foreign fragments On the vector, there is no need to judge the insertion direction of the gene, which simplifies the experimental steps. Since this idea can be used in all PCR amplification and vector construction, this method is suitable for cloning PCR products amplified by all current DNA polymerases, especially suitable for PCR with the polymerase (Pfu) with the best fidelity at present. Amplification minimizes mutations introduced by PCR and saves the trouble of sequencing. In order to avoid the background interference of the self-ligation of the vector, an expression unit of a lethal gene is also introduced into the vector, which truly achieves zero background cloning. It can perform fast connection in 5 minutes and fast transformation in 30 minutes, which greatly improves the experimental efficiency. The entire gene cloning work can be completed within one day, and the cloning recombination efficiency can theoretically reach more than 99%.

2. Compatible with all PCR-based library construction methods reported so far, PCR products can be used to directly construct expression libraries, which is not only suitable for high-throughput cloning and expression using bacteria and fungi as hosts, but also suitable for animals, plants, etc. As a host for high-throughput cloning and expression.

3. The experiment cost is low. First of all, this technology eliminates the two steps of judging the insertion direction of the gene by PCR amplification and sequencing the correctness of the gene, which greatly reduces the cost of the experiment; secondly, it does not use Cre enzyme and topoisomerase carrier, only conventional T ₄ DNA polymerase and ligase, the cost of each reaction is about RMB 1, which is a very economical method. If high-throughput cloning and expression are performed, its advantages will be more prominent.

Detailed ways

Below with embodiment the present invention is further described:

Example 1:

This method was used to express the oligo-1,6-glucosidase of B. subtilis in E. coli.

Select expression vector pBV220 and target gene oligo-1,6-glucosidase. Design a polynucleotide linker, introduce a Cpo I and Not I restriction site at both ends, connect a lethal gene expression unit in the middle, introduce this linker into the E. coli expression vector pBV220, and obtain zero background high pass Quantify the expression vector for directional cloning, digest the vector with Cpo I and Not I, and recover a large fragment, each of which has a sticky end with 3 or 4 bases protruding from its 5' end. According to the gene sequence of Bacillus subtilis oligomerization-1,6-glucosidase, a pair of primers were designed, and several extra nucleotides of 5'GACT3' and 5'GGCCT3' were introduced at the 5' end respectively; The genomic DNA of Bacillus was used as a template, and high-fidelity DNA polymerase (Pfu) was used for PCR amplification. In the presence of dATP, the obtained product was treated with T ₄ DNA polymerase, and there was a 5' at both ends of the PCR product. The cohesive end with 3 or 4 bases protruding from the end, the product can be directional cloned into the vector cut by Cpo I and Not I, and then transformed into Escherichia coli, more than 99% of the obtained transformants can correctly express oligomeric - 1,6-glucosidase.

Example 2:

Expression of Aspergillus niger endo-inulinase in Pichia pastoris.

Design the same linker according to the method of Example 1, introduce the Pichia pastoris expression vector pIC3.5K, design the primers for amplifying the endo-inulinase gene of Aspergillus niger by the same method, and amplify the endo-inulinase gene of Aspergillus niger with Pfu polymerase Powder enzyme gene, the amplified product was treated with _T4 DNA polymerase in the presence of dATP, and then directionally cloned into the vector cut by Cpo I and Not I, and transformed into Pichia pastoris, and the obtained transformant was 90 More than % can correctly express the endo-inulinase.

Claims (5)

1, the method for a kind of zero background, high-throughput, directed cloning expression, mainly comprise an expression vector and through the goal gene of PCR primer amplification, it is characterized in that in expression vector, having introduced two particular restriction endonuclease digestion site sequences and a lethal gene is expressed the unit; 5 ' end in amplification target gene one couple of PCR primers has been introduced 2～5 specific deoxynucleotides respectively, and under the condition of dATP or dGTP or dCTP or dTTP existence, amplified production is through T ₄The sticky end that archaeal dna polymerase digestion back produces mates with the sticky end that the carrier double digestion obtains, thus can directed cloning on the carrier of two special restriction enzymes double zyme cuttings.

2, the method for zero background according to claim 1, high-throughput, directed cloning expression, it is characterized in that on expression vector, at first designing the joint of polynucleotide, its two ends are introduced a CpoI and NotI restriction enzyme site respectively, the expression unit of a lethal gene of indirect, this joint is introduced on the expression vector, carrier produces such two sticky ends through CpoI and NotI double digestion:

3′GC——————CGCCGG 5′

5′GTCCG——————GC 3′；

5 of goal gene one couple of PCR primers ' end is introduced 5 ' GACT3 ' and 5 ' GGCCT3 ' 3--4 Nucleotide so respectively, and the product that pcr amplification obtains adds dATP and T ₄The archaeal dna polymerase effect produces such sticky end:

5′GACT————A 3′

3′A——————TCCGG?5′；

This sticky end just in time can with the carrier double digestion produce the sticky end coupling, thereby make PCR product directed cloning to carrier.

3, the method for zero background according to claim 1, high-throughput, directed cloning expression, two special restriction enzymes that it is characterized in that polynucleotide joint two ends, can be enzyme cut the back getable 5 ' the base kind of outstanding sticky end is no more than 3 kinds restriction enzyme, concrete restriction enzyme can be any two kinds or CpoI, EarI, among SapI, the StyI any among CpoI, EarI, NotI, SapI, the StyI.

4, the method expressed of zero background according to claim 1, high-throughput, directed cloning, it is characterized in that can to build the method in storehouse compatible with all PCR-based.

5, the method expressed of zero background according to claim 1, high-throughput, directed cloning is characterized in that can be used for high-throughout clone and the expression as the host of bacterium, fungi, animal, plant.