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yeast plasmid vector

Types of gene expression vectors

synonym Shuttle carrier (Shuttle carrier) generally refers to the carrier of yeast matrix

This entry is made by Compilation and application of scientific encyclopedia entries of "Science Popularization China" to examine.

The yeast plasmid vector is Gene expression vector One of the Escherichia coli They can be replicated and amplified in yeast system, so they are also called shuttle vectors. It is divided into two categories: integration carrier and self replication carrier. ① Integration vector: it contains a yeast URA3 marker gene and replication and reporting gene of Escherichia coli. ② Self replicating vector: This kind of vector can self replicate in yeast.

Chinese name: yeast plasmid vector
Foreign name: Yeast plasmid vector
Essence: carrier

Alias: Shuttle carrier
Classification: Integration carrier and self replication carrier
Features: E. coli and yeast system can be replicated and expanded

catalog

one brief introduction
two Characteristics of yeast plasmid vector
three Carrier technology

brief introduction

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The yeast plasmid vector is Gene expression vector One of the Escherichia coli It can be replicated and amplified in yeast system, so it is also called Shuttle carrier 。 It is divided into two categories: integration carrier and self replication carrier. ① Integration vector: it contains a yeast URA3 marker gene and replication and reporting genes of Escherichia coli. Due to homologous recombination between plasmid DNA and yeast genomic DNA, plasmid integration and replication can be detected in transformed cells. The YIp carrier (yeast integrating plasmid) in the integration carrier has low conversion efficiency and is unstable; ② Self replicating vector: This kind of vector can self replicate in yeast, mainly including YRp (yeast replication plasma), YEp (yeast extrachromosomal plasma) and YCp (yeast centromer plasma). YRp is very unstable in genetics; YEp can be quickly recombined with yeast endogenous plasmid, and the recombinant YEp vector can be quickly replicated and amplified. YCp plasmid is characterized by low copy number and stable heredity; ③ Saccharomyces cerevisiae carrier system: Saccharomyces cerevisiae In the operation of genetic engineering technology, as a clone host or as a regulatory expression sequence (such as RNA polymerase recognition site and ribosome recognition site) Cloning vector , which is widely used. Some self replicating plasmids (such as E. coli plasmid) can also obtain high copies in the cell nucleus of yeast. For example, the maximum expression of genes in yeast requires specific promoters, such as the promoters of genes such as alcohol dehydrogenase, isozyme-1, phosphoglycerol kinase, acid phosphatase and alpha factor. adopt DNA recombination technology It can also use Saccharomyces cerevisiae to produce foreign proteins. hepatitis B vaccine It is the first medical protein obtained by using yeast as the host. In addition, protein products such as tissue type plasminogen activator, insulin and hirudin are also produced by using yeast as the host cell.

Due to Saccharomyces cerevisiae Eukaryotic introns cannot be identified and processed, so foreign genes must use cDNA. It is also very important whether the product protein is expressed in cells or secreted outward, because this is related to the process design of downstream genetic engineering. In addition, proper promoters and terminators should be selected when using the expression system of Saccharomyces cerevisiae; Check the stability of the expression cassette; According to the different accumulation sites of foreign proteins, corresponding treatment steps were taken; Pay attention to increasing output^[1] 。

Characteristics of yeast plasmid vector

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Yeast plasmid vector can be used in Escherichia coli Replication and amplification can also be replicated and amplified in yeast system, so this kind of vector is also called shuttlevector.

In genetic engineering, the above viral expression vector is used to Mammalian cell It is very practical to carry out medium - and small-scale expression. It is also necessary to be familiar with the tissue culture technology. but Viral vector There are still technical difficulties, time consuming and uneconomical problems in the application of the. Between bacterial plasmid and Animal virus The yeast plasmid vector of "Vectors of News", from the technical and economic point of view, may be more suitable for the needs of most domestic research laboratories at present.

according to Transformed cell The yeast plasmid vector can be divided into two basic types, namely integration vector and self replication vector. The following criteria must be carefully considered when selecting yeast carrier: ① Escherichia coli And yeast have appropriate genetic markers; ② Considering the actual needs, consider the replication mode in yeast; ③ Copy number in yeast and Escherichia coli; ④ There should be an easy way to filter inserts.

Carrier technology

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Gene expression vector The construction of the target gene (that is, the combination of the target gene and the carrier) is the second step of the implementation of genetic engineering, and is also the core of genetic engineering.

The process of combining the target gene with the carrier is actually the process of recombination of DNA from different sources. The so-called vector is a DNA fragment that connects the target gene and can be copied independently of the cell chromosome. At present, bacterial plasmid vector λ bacteriophage vector Cox plasmid vector, virus vector, etc.

An ideal Gene vector It should have the following basic conditions: ① It can host cell The independent and stable self replication of DNA and the insertion of foreign genes into its DNA still maintain a stable replication state and genetic characteristics; ② It is easy to isolate and purify from host cells; ③ In its DNA sequence Restriction endonuclease Site (preferably single Restriction site )。 These sites are located in the non essential area of DNA replication, and foreign DNA can be inserted into these sites, but do not affect the replication of the vector's own DNA; ④ With observable phenotypic characteristics, these characteristics can be used as Recombinant DNA Selection flag for.

The above basic conditions are mainly derived from prokaryotes DNA recombination From. At the eukaryotic stage, the method of introducing exogenous DNA into the host has been improved, breaking through the original requirements Recombinant DNA Therefore, the ability of exogenous DNA to enter the host is enhanced; At the same time, due to the improvement of screening recombinant technology, the original requirement for selective markers on vectors or foreign DNA has been replaced by hybridization technology, immunology technology, etc., thus expanding the DNA recombination technology Range of use. Therefore, the most basic requirements for carriers have changed, that is, independent replication capability and available restriction enzyme cut points.

(1) Plasmid vector

Plasmids are small circular DNA molecule 1~200kb (1kb=1000 base pairs). Plasmids are different from viruses. They are bare DNA molecules without coat proteins. In the genome, there is no lysozyme gene. Plasmids in host cell At the same time, some non chromosome controlled genetic traits encoded are expressed, giving host cells some additional characteristics, including resistance characteristics, metabolic characteristics, etc. Resistance to antibiotics is one of the most important coding characteristics of plasmids.

The vast majority of plasmid vectors are based on natural plasmids and artificially modified and constructed. An ideal bacterial plasmid vector, in addition to the same characteristics of other types of vectors, should also have the following conditions: ① the relative molecular weight should be as small as possible, the smaller the plasmid, the higher the copy number, and easy to extract and purify; ② The DNA structure and function are clear, and the plasmid sequence contains a series of single restriction enzyme digestion sites Polyclonal site To facilitate the insertion of foreign fragments with different ends; ③ It has one or more appropriate selection markers in the host to screen clones carrying targeted fragments. Such selection markers can be divided into dominant markers and nutrient deficiency markers. The most important dominant markers are various antibiotic resistance genes (such as ampicillin resistance gene, tetracycline resistance gene, aporamycin resistance gene, chloramphenicol resistance gene, etc.); ④ The flowing gene is deleted so that the plasmid will not be transferred from one bacterium to another.

In genetic engineering, some vectors can be used for special purposes such as the expression of foreign genes, but general cloning experiments can be divided into several different types according to the different properties of vectors. if DNA recombination In order to obtain a large number of high-purity DNA fragments, plasmid vectors with high copy numbers can be selected, such as relaxed replicon plasmids such as ColE1; Some foreign genes were cloned with high copy number plasmid vector. If the product content is too high, it will interfere with the metabolic activities of the host cell. For such cloned genes, it is better to use plasmid vectors derived from the tight replicon PSC101, such as PLG338, PLG339, etc. These plasmids have only a few copies in each cell, which can reproduce cloned foreign DNA fragments at a low level of gene dose.

plasmid Cloning vector The following principles should be followed in the design and construction process of.

① Select appropriate starting plasmid

The starting plasmid is also called the parent plasmid, which should contain the necessary elements of the plasmid cloning vector, such as the replication start site, the selection of marker genes, the cloning site, the promoter and the terminator.

② Correct acquisition of elements for constructing plasmid cloning vector

Generally adopted Restriction endonuclease Cut out plasmid DNA molecule DNA fragments containing certain elements can be obtained, and specific DNA fragments containing certain elements can also be amplified from target DNA by PCR technology.

③ Assemble appropriate selective marker genes

What kind of selective marker genes should be assembled in the constructed plasmid cloning vector must be based on the Receptor cell Is determined by the characteristics of.

④ Select the appropriate promoter

In order to construct the expression plasmid Cloning vector The appropriate promoter must be assembled. When the gene of the designed eukaryote must be expressed in the prokaryote, the promoter of the prokaryote or virus (bacteriophage) gene is often used instead Eukaryotic cell The promoter of prokaryotic gene can still be used when it is expressed in.

Since plasmid vectors can be used to prepare some cloned DNA fragments easily, quickly and in large quantities, they have been widely used and improved over the years, producing more and more useful plasmid vectors, such as PBR322, PUC series, PSP series, BluescriptM, etc. Most of these carriers have been commercialized. It can be purchased directly.

(2) Viruses and Bacteriophage carrier

The virus is mainly composed of DNA (or RNA) and coat protein, and becomes virus particles after packaging. Through infection, virus particles enter host cell The host cell synthesis system is used for DNA (or RNA) replication and shell protein synthesis to achieve the proliferation of virus particles. The virus infected with bacteria is specially called bacteriophage, and thus constructed Cloning vector It is called phage cloning vector.

Phage, as a vector, can insert some foreign DNA fragments 10~20kb long or even larger, and because of its high proliferation ability, it is conducive to the amplification of the target gene, so it becomes one of the important vectors for current genetic engineering research.

Wild type phages must be modified to become ideal vectors for genetic engineering. The first phage to be transformed into a carrier is Lambda bacteriophage , plus Single strand bacteriophage Vector, viscous plasmid vector, etc. The λ bacteriophage consists of a head and a tail. Its genome is a linear double stranded DNA molecule about 49kb long, assembled inside the head protein shell, and its sequence has been completely measured. When λ bacteriophage is infected, genomic DNA is injected into Escherichia coli through tail tube, while its protein shell is left outside the bacterium. DNA entry Escherichia coli Then, the 12 bp complementary single chain at its two ends is cyclized to form a circular double chain, which can be propagated in two different ways: the lysogenic way and the lysogenic way. The λ phage contains a linear double stranded DNA molecule with a length of 48502 bp. The two segments each have complementary sticky ends protruding from the 5 'end of 12 nucleotides, when λ DNA enters host cell Then, the complementary viscous ends are connected into a ring DNA molecule , the connection is called cos Locus.

structure λ bacteriophage vector The basic approaches of the restriction enzyme are as follows: ① erase some recognition sequences of a restriction enzyme on λ DNA molecule, and only retain 1~2 recognition sequences in the non essential region; ② Use the appropriate Restriction endonuclease Cut off some unnecessary areas, but the λ DNA vector constructed from this should not be less than 38kb; ③ At λ DNA molecule A selectable marker gene was inserted into the appropriate region of. It is worth pointing out that there is no universal λ bacteriophage vector suitable for cloning all DNA fragments, and appropriate bacteriophage vector must be selected according to needs^[2] 。

(3) Artificial chromosome Cloning vector

The artificial chromosome cloning vector is actually a shuttle cloning vector, which contains the first receptor (Escherichia coli) source plasmid replication start site necessary for plasmid cloning vector, as well as the second receptor (such as yeast) Chromosome DNA The sequence of centromere, telomere and replication start site, as well as appropriate selective marker genes. Such cloning vector Receptor cell High copy replication can be carried out in the form of plasmid replication. After recombination with the target DNA fragment in vitro, the second receptor cell can be transformed.

Press Chromosome DNA The form of replication is used for replication and transmission, and the clone of the first receptor is screened, generally using antibiotic resistance selection markers; However, screening the clone of the second receptor is often complementary to the receptor Auxotrophic type 。 And others Cloning vector comparison, Artificial chromosome The characteristic of cloning vector is that it can accommodate foreign DNA fragments up to 1000 kb or even 3000 kb.

Common artificial chromosome cloning vectors mainly include Yeast artificial chromosome 、 Bacterial artificial chromosome Vector and P1 artificial chromosome carrier, etc.

Yeast artificial staining carrier (YAC) is a kind of carrier Saccharomyces cerevisiae The working environment of the vector constructed by the replication element of the chromosome of Saccharomyces cerevisiae is also in Saccharomyces cerevisiae. The shape of Saccharomyces cerevisiae is oblate and oval, and its generation time is 90 min. The replication element of YAC vector is its core component. The essential elements for replication in yeast include the replication starting sequence, i.e. autonomous replication sequence, centromere for mitosis and meiosis and two telomeres (TEL). The selective marker of YAC vector mainly uses nutrient deficient genes, such as tryptophan, leucine and histidine synthesis deficient genes trpl 、 leu2 Astringency and harmony his3 Uracil synthesis deficient gene ura3 And ocher mutation suppressor gene sup4 Etc. Host yeast (such as AB1380) working with YAC carrier Thymine Synthetic gene with an ochre mutation Ade2-1 。 The yeast with this mutation Basic medium The red colony is formed on the sup4 When the carrier of Ade2-1 Gene mutation effect, forming normal white colonies.

This phenomenon of colony color change can be used to screen recombinants containing exogenous DNA fragments inserted into the vector.

Bacterial artificial chromosome The carrier (BAC) is based on Escherichia coli High throughput low copy plasmid vector constructed from F plasmid. Each ring DNA molecule It carries an antibiotic resistance marker, a strictly controlled replicon oriS derived from Escherichia coli F factor (fertility factor), and an ATP driven helicase that is easy to replicate DNA. The low copy of BAC vector can avoid the generation of chimeras and reduce the impact of foreign gene expression products on host cell Toxic and side effects. The new BAC vector can screen the recombinants containing inserted fragments through the principle of alpha complementation, and has designed the Not enzyme digestion site for recovering cloned DNA and for cloning DNA sequencing Sp6 promoter, T7 promoter. Not I recognition sequence, the site is very rare. After the recombinant is digested by Not I, a complete insertion fragment can be obtained. Sp6 and T7 are promoters from bacteriophages, which are used for sequencing the ends of inserted fragments^[3] 。

P1 artificial chromosome vector (PAC) combines the best characteristics of P1 vector and BAC vector, including positive selection marker sacB and plasmid replicon and lysis replicon of phage P1. However, in addition to packaging the connecting product into Lambda bacteriophage Granules and use at cre loxP site Site-specific recombination In addition to producing plasmid molecules, the circular recombinant PAC produced in the process of carrier connection may also be introduced by electroporation Escherichia coli And maintained in a single copy plasmid state. PAC based human Genome Library The size of the inserted fragment is between 60 and 150 kb.

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