Homologous recombinant vector for mammary gland specific expression of anti-k 88ac antigen antibody gene and application thereof
阅读说明：本技术 乳腺特异性表达抗k88ac抗原抗体基因的同源重组载体及应用 (Homologous recombinant vector for mammary gland specific expression of anti-k 88ac antigen antibody gene and application thereof ) 是由 李秋艳 林如涛 李志远 郝海阳 史文姝 黄彤彤 牛建芹 李梦然 于 2019-10-09 设计创作，主要内容包括：本发明提供一种乳腺特异性表达抗k88ac抗原抗体基因的同源重组载体及应用。所述同源重组载体是以真核表达载体为骨架载体,同源重组载体中至少包括正筛选元件、负筛选标记基因、抗k88ac抗原抗体基因表达盒以及rosa26基因的同源左臂和同源右臂；其中,正筛选元件包括顺次连接的重组酶识别序列-正筛选标记基因-重组酶基因-重组酶识别序列。利用所述同源重组载体转染哺乳动物体细胞,获得的阳性细胞rosa26基因第一个内含子定点整合抗k88ac抗原抗体基因；利用本发明方法成功获得了乳腺特异性表达抗k88ac抗原抗体的转基因猪细胞系,为通过转基因技术制备抗腹泻转基因猪奠定基础。(The invention provides a homologous recombinant vector for specifically expressing an anti-k 88ac antigen antibody gene in mammary gland and application thereof. The homologous recombination vector takes a eukaryotic expression vector as a skeleton vector, and at least comprises a positive screening element, a negative screening marker gene, an anti-k 88ac antigen antibody gene expression box, and a homologous left arm and a homologous right arm of a rosa26 gene; wherein the positive screening element comprises a recombinase recognition sequence-positive screening marker gene-recombinase recognition sequence which are connected in sequence. Transfecting mammalian somatic cells by using the homologous recombination vector to obtain a positive cell rosa26 gene, wherein the anti-k 88ac antigen antibody gene is integrated into the first intron of the gene at a fixed point; the method successfully obtains the transgenic pig cell line of the mammary gland specificity expression anti-k 88ac antigen antibody, and lays a foundation for preparing the anti-diarrhea transgenic pig by a transgenic technology.)
1. The homologous recombination vector for specifically expressing the anti-k 88ac antigen-antibody gene in the mammary gland is characterized in that the homologous recombination vector takes a eukaryotic expression vector as a skeleton vector, and at least comprises a positive screening element, a negative screening marker gene, an anti-k 88ac antigen-antibody gene expression box, and a homologous left arm and a homologous right arm of a rosa26 gene;
wherein the positive screening element comprises a recombinase recognition sequence-positive screening marker gene-recombinase recognition sequence connected in sequence;
one end of the positive screening element is connected with the homologous left arm, and the other end of the positive screening element is connected with the anti-k 88ac antigen antibody gene expression cassette; the other end of the anti-k 88ac antigen antibody gene expression cassette is connected with the homologous right arm, and the outer side of the homologous right arm is connected with a negative selection marker gene.
2. The homologous recombinant vector according to claim 1, wherein the positive selection marker gene comprises a gene encoding neomycin phosphotransferase, hygromycin B phosphotransferase, hypoxanthine phosphoribosyltransferase or puromycin acetyltransferase; the negative selection marker gene comprises a gene encoding diphtheria toxin or thymidine; the recombinase gene includes a gene encoding Cre enzyme or Flp enzyme.
3. The homologous recombination vector of claim 2, wherein the positive selection element is Loxp-OCT4 promoter-Cre-PGK-Neo-Loxp.
4. The homologous recombinant vector according to claim 1, wherein the anti-k 88ac antigen antibody gene expression cassette is human lactoferrin hLF promoter-anti-k 88ac antigen antibody gene-hLF terminator.
5. The homologous recombination vector according to claim 1, wherein said homologous recombination vector comprises the following elements linked in sequence: homologous left arm-Loxp-OCT 4 promoter-Cre-PGK-Neo-Loxp-human lactoferrin hLF promoter-anti-k 88ac antigen antibody gene-hLF terminator-homologous right arm-PGK-dTA.
6. The homologous recombination vector of claim 5, wherein the homologous left arm and the homologous right arm are the homologous left arm and the homologous right arm from the porcine rosa26 gene, and the nucleotide sequences thereof are shown in SEQ ID NO 1 and 2, respectively; the nucleotide sequence of the anti-k 88ac antigen antibody gene is shown in SEQ ID NO. 3.
7. The homologous recombination vector of claim 1, wherein the nucleotide sequence of the homologous recombination vector is represented by SEQ ID NO. 4.
8. A cell line specifically expressing anti-k 88ac antigen antibody gene in mammary gland, characterized in that, the homologous recombination vector of any one of claims 1-7 is used to transfect mammalian somatic cells to obtain the target-positive cell clone, namely the cell line specifically expressing anti-k 88ac antigen antibody gene in mammary gland.
9. Use of the homologous recombination vector of any one of claims 1 to 7 or the cell line of claim 8 for the preparation of a transgenic animal specifically expressing an anti-k 88ac antigen antibody in mammary glands, wherein the animal comprises a pig.
10. Use according to claim 9, characterized in that the homologous recombination vector is transfected into the same porcine cell line as the source of the DNA of its homology arms.
Technical Field
The invention belongs to the field of animal genetic engineering, and particularly relates to a mammary gland specificity expression homologous recombinant vector of an anti-k 88ac antigen antibody gene and application thereof.
Background
The diarrhea of piglets is the first in the harm of the pig industry, seriously threatens the healthy development of the pig industry and is a typical multifactorial disease under the intensive pig-raising production condition. However, the infectious diarrhea is common to the suckling piglets, and especially the diarrhea of the piglets caused by the Escherichia coli carrying the k88ac pilin is the most common.
Escherichia coli k88ac pilin belongs to one of Escherichia coli cilia antigens, is protein, is thermolabile, is located on the surface of thallus, and has close relation with enteropathogenesis of bacterial strain. Escherichia coli k88ac is a main pathogen causing piglet diarrhea, and Escherichia coli k88ac pilin acts as an adhesion factor in a pig body, so that Escherichia coli is fixed on mucosa villi of the front intestinal tract to generate enterotoxin, and common piglet diarrhea is caused.
However, at present, no good monoclonal antibody for k88ac escherichia coli is used for resisting the harm of k88ac escherichia coli to piglets, and the diarrhea disease of the piglets is still a big reason for threatening the pig industry. So far, no report of a single-chain antibody which can specifically bind to Escherichia coli k88ac and effectively exert immune resistance is found in China. Therefore, the inventor obtains the single-chain antibody of the anti-k 88ac escherichia coli by a biotechnology means, and simultaneously has a remarkable bacteriostatic effect, and the aims of preparing the anti-diarrhea transgenic pig by a transgenic technology and reducing the diarrhea incidence of piglets in China are prompted.
Disclosure of Invention
The invention aims to provide a mammary gland specificity expression anti-k 88ac antigen antibody gene homologous recombinant vector and application thereof.
The invention has the following conception: the anti-k 88ac antigen-antibody gene is transferred into pig fibroblasts by a genetic engineering means (by constructing a homologous recombinant vector of the mammary gland specificity expression anti-k 88ac antigen-antibody gene), and is specifically expressed in mammary glands, so that the content of the anti-k 88ac antigen-antibody in milk is increased, and the aim of reducing the diarrhea incidence of piglets is fulfilled for the piglets.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a homologous recombination vector for mammary gland specific expression of an anti-k 88ac antigen antibody gene, wherein the homologous recombination vector takes a eukaryotic expression vector as a skeleton vector, and at least comprises a positive screening element, a negative screening marker gene, an anti-k 88ac antigen antibody gene expression cassette and a homologous left arm and a homologous right arm of a rosa26 gene.
Preferably, the homologous recombination vector is a vector BAC as a backbone vector.
Wherein the positive screening element comprises a recombinase recognition sequence-positive screening marker gene-recombinase recognition sequence which are connected in sequence.
One end of the positive screening element is connected with the homologous left arm, and the other end of the positive screening element is connected with the anti-k 88ac antigen antibody gene expression cassette; the other end of the anti-k 88ac antigen antibody gene expression cassette is connected with the homologous right arm, and the outer side of the homologous right arm is connected with a negative selection marker gene.
Preferably, the positive selection marker gene includes, but is not limited to, a gene encoding neomycin phosphotransferase (Neo), hygromycin B phosphotransferase, hypoxanthine phosphoribosyl transferase, or puromycin acetyltransferase.
Preferably, the negative selection marker gene includes, but is not limited to, a gene encoding diphtheria toxin or thymidine (dTA).
The recombinase gene of the invention includes a gene encoding Cre enzyme or Flp enzyme.
Preferably, the positive screening element in the homologous recombination vector is Loxp-OCT4 promoter-Cre-PGK-Neo-Loxp. Wherein, the Cre expression frame is regulated and controlled by an embryonic stage
Preferably, the anti-k 88ac antigen-antibody gene expression cassette is human lactoferrin hLF promoter-anti-k 88ac antigen-antibody gene-hLF terminator.
More preferably, the homologous recombination vector comprises the following elements connected in sequence: homologous left arm-Loxp-
Wherein the homologous left arm is the homologous left arm from the porcine rosa26 gene, preferably at least 1.1kb sequence from
The nucleotide sequence of the anti-k 88ac antigen antibody gene is shown in SEQ ID NO:3, respectively.
In one embodiment of the invention, the homologous recombinant vector for mammary gland specific expression of the anti-K88 ac antigen antibody gene is pHS-AVC-YW079HLF-K88 (figure 1), wherein O represents a specific promoter of porcine early embryonic development
The full length of the nucleotide sequence of the homologous recombinant vector pHS-AVC-YW079HLF-K88 is shown as SEQ ID NO:4, respectively.
In a second aspect, the invention provides a cell line specifically expressing an anti-k 88ac antigen antibody gene in mammary gland, and the homologous recombination vector is used to transfect mammalian somatic cells to obtain a target-positive cell clone, namely the cell line specifically expressing the anti-k 88ac antigen antibody gene in mammary gland.
In a third aspect, the invention provides the use of the homologous recombination vector or the cell line for preparing a transgenic animal specifically expressing the anti-k 88ac antigen antibody in mammary gland, wherein the animal comprises a pig.
In the application, the homologous recombination vector is transfected into a pig cell line with the same DNA source of the homologous arm.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the homologous recombination vector of the mammary gland specificity expression anti-k 88ac antigen antibody gene is constructed for the first time, and the transgenic pig cell line of the mammary gland specificity expression anti-k 88ac antigen antibody is successfully obtained, so that a foundation is laid for preparing the anti-diarrhea transgenic pig by a transgenic technology.
Drawings
FIG. 1 is a plasmid map of the homologous recombinant vector pHS-AVC-YW079HLF-K88 of the present invention.
FIG. 2 shows the results of the identification of the homologous recombination between the short arm (A) and the long arm (B) in example 2 of the present invention. Wherein 1300bp is a short-arm positive fragment. 8000bp is a long-arm positive fragment.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.