阅读说明：本技术 一种组成型乳酸菌启动子、重组载体及其重组菌和应用 (Constitutive lactobacillus promoter, recombinant vector, recombinant bacterium and application thereof ) 是由 金万洙 于 2019-04-11 设计创作，主要内容包括：本发明提供了一种组成型乳酸菌启动子、重组载体及其重组菌和应用,属于基因工程领域。本发明提供的启动子P<Sub>gapA</Sub>的核苷酸序列如SEQ ID No.1所示。所述启动子P<Sub>gapA</Sub>能够启动下游基因在乳酸菌中的高效表达,蛋白表达活性显著高于传统诱导型P<Sub>NisinA</Sub>的表达活性,是一种超高效启动子。本发明的启动子PgapA与目标基因其所组成的蛋白表达框,表达载体以及宿主菌可以用于肽类和蛋白类药物的口服药物递送,不仅在基因工程中具有良好的应用前景,还可以提高患者的服药依从性,有望产生可观的社会和经济效益。(The invention provides a constitutive lactobacillus promoter, a recombinant vector, recombinant bacteria and application thereof, and belongs to the field of genetic engineering. The promoter P provided by the invention gapA The nucleotide sequence of (A) is shown in SEQ ID No. 1. The promoter P gapA Can start the high-efficiency expression of downstream genes in lactic acid bacteria, and the protein expression activity is obviously higher than that of the traditional inducible P NisinA Is an ultra-efficient promoter. Promoters of the inventionThe protein expression frame, the expression vector and the host bacterium formed by PgapA and the target gene can be used for oral drug delivery of peptide and protein drugs, and the protein expression frame, the expression vector and the host bacterium have good application prospects in genetic engineering, can improve the drug administration compliance of patients, and are expected to generate considerable social and economic benefits.)

1. The constitutive lactobacillus promoter is characterized in that the nucleotide sequence of the promoter is shown in a sequence table SEQ ID No.1 or is a complementary sequence shown in the sequence table SEQ ID No. 1.

2. A recombinant vector comprising the promoter of claim 1.

3. The recombinant vector according to claim 2, wherein the basic vector in the recombinant vector is pNZ 8149;

the recombinant vector further comprises a target gene;

the target genes are sequences encoding inhibitors of the human PD-1/PD-L1 signaling pathway and sequences encoding inhibitors of the PD-1/PD-L1 signaling pathway and the TGF beta/TGF beta R2 signaling pathway.

4. The recombinant vector according to claim 3, wherein the sequence encoding the inhibitor of the human PD-1/PD-L1 signaling pathway comprises a nucleotide sequence of a human PD-1 ectodomain structural protein or a sequence encoding a fusion protein comprising a human PD-1 ectodomain structural protein;

the nucleotide sequence of the human PD-1 ectodomain structural protein is shown in SEQ ID No. 2;

the sequence for encoding the fusion protein containing the human PD-1 ectodomain structural protein is a sequence for encoding human PD-1 ectodomain-human TGF beta R2 ectodomain fusion protein.

5. The recombinant vector according to claim 3, wherein the sequences encoding inhibitors of the PD-1/PD-L1 signaling pathway and the TGF β/TGF β R2 signaling pathway are sequences encoding human PD-1 ectodomain-human TGF β R2 ectodomain fusion proteins.

6. The recombinant vector according to claim 4 or 5, wherein the sequence encoding the fusion protein of human PD-1 ectodomain-human TGF β R2 ectodomain is shown as SEQ ID No. 3.

7. A recombinant cell comprising the promoter of claim 1 or the recombinant vector of any one of claims 2 to 6.

8. The recombinant cell of claim 7, wherein the basal cell of the recombinant cell comprises a normal gut bacterium;

the normal intestinal bacteria include Bifidobacterium, Lactobacillus, or Streptococcus.

9. Use of the promoter according to claim 1, the recombinant vector according to any one of claims 2 to 6 or the recombinant cell according to claim 7 or 8 for expressing a gene or an anabolic product of interest.

10. Use of the promoter of claim 1, the recombinant vector of any one of claims 2 to 6, or the recombinant cell of claim 7 or 8 for the preparation of a protein or polypeptide drug.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a constitutive lactobacillus promoter, a recombinant vector, recombinant bacteria and application thereof.

Background

With the development of biotechnology, many active polypeptides and proteins are developed into drugs and applied clinically. Drugs such as polypeptides, proteins, enzymes, hormones, vaccines, cell growth factors, and monoclonal antibodies for therapeutic or diagnostic use are continuously emerging, and nearly hundreds of polypeptide and protein drugs represented by erythropoietin, interferon, and insulin have been clinically used. Compared with the traditional chemical synthesis medicines, the polypeptide and protein medicines have the advantages of specific action target, definite in-vivo action, strong drug effect, little toxic and side effect and the like, so the polypeptide and protein medicines are increasingly widely applied to the prevention and treatment of modern diseases. However, polypeptides and proteins such as antibodies for immunotherapy have large molecular weight, relatively complex structure and special physicochemical properties, so that the body generates various barriers such as acid barrier, enzyme barrier and membrane barrier to the polypeptides and proteins, and the oral absorption of the drugs is limited. Therefore, at present, such drugs are still limited to invasive administration modes such as intravenous, subcutaneous and intramuscular injection, which results in high production, transportation and storage costs and risks of disease infection, and is a big disadvantage.

In recent years, with the continuous development of bacterial genomics and the continuous abundance of genetic manipulation tools, drug delivery using live probiotics as a delivery carrier is gradually a hot spot of application research. Compared to traditional antibody drug delivery systems, probiotic oral drug delivery systems have the following advantages: the probiotics is widely applied in the food field, and has low acquisition cost, high safety and no toxic or side effect; secondly, the liquid or solid microbial inoculum prepared by the recombinant probiotics can be directly orally taken without injection, so that adverse reaction of an injection part is avoided; and thirdly, the oral delivery system cannot generate drug resistance, so that the probiotics is a very good delivery carrier of polypeptide and protein drugs. However, the prior art has the problem of low protein expression efficiency, and the application of the system is greatly limited.

The promoter is a key DNA sequence for RNA polymerase to recognize, combine and start transcription, and has important regulation and control effect on gene expression. Researches show that the influence of the promoter in various sequences for controlling gene expression is over 45 percent, so that the screening of the high-activity promoter is important for improving the gene expression and realizing the clinical application of an oral drug delivery system taking probiotics as a carrier. At present, the promoter in the probiotics has the problem of starting performance, and the effect of the probiotics delivery vector on treating diseases is influenced.

Disclosure of Invention

In view of the above, the invention provides an ultra-efficient constitutive lactic acid bacteria promoter to solve the problem of low protein expression level in the existing lactic acid bacteria system. Meanwhile, based on the constitutive lactobacillus promoter, the invention also provides a recombinant vector containing the promoter, recombinant bacteria containing the promoter and application of the recombinant vector, wherein the recombinant vector or the recombinant bacteria can be used for oral drug delivery of protein drugs and can be used for preparing drugs for treating various diseases based on efficient downstream gene expression.

The invention provides a constitutive lactobacillus promoter, wherein the nucleotide sequence of the promoter is shown as a sequence table SEQ ID No.1 or a complementary sequence shown as a sequence table SEQ ID No. 1.

The invention provides a recombinant vector containing the promoter.

Preferably, the basic vector in the recombinant vector is pNZ 8149;

the recombinant vector further comprises a target gene;

the target genes are sequences encoding inhibitors of the human PD-1/PD-L1 signaling pathway and sequences encoding inhibitors of the PD-1/PD-L1 signaling pathway and the TGF beta/TGF beta R2 signaling pathway.

Preferably, the sequence encoding the inhibitor of the human PD-1/PD-L1 signaling pathway comprises a nucleotide sequence of a human PD-1 ectodomain structural protein or a sequence encoding a fusion protein comprising a human PD-1 ectodomain structural protein;

the nucleotide sequence of the human PD-1 ectodomain structural protein is shown in SEQ ID No. 2;

the sequence for encoding the fusion protein containing the human PD-1 ectodomain structural protein is a sequence for encoding human PD-1 ectodomain-human TGF beta R2 ectodomain fusion protein.

Preferably, the sequence encoding the inhibitor for inhibiting the PD-1/PD-L1 signaling pathway and the TGF beta/TGF beta R2 signaling pathway is a sequence encoding a human PD-1 ectodomain-human TGF beta R2 ectodomain fusion protein.

Preferably, the sequence encoding the fusion protein of human PD-1 ectodomain-human TGF beta R2 ectodomain is shown in SEQ ID No. 3.

The present invention provides a recombinant cell comprising preferably said promoter or preferably said recombinant vector.

Preferably, the basal cells in the recombinant cells comprise intestinal normal bacteria;

the normal intestinal bacteria include Bifidobacterium, Lactobacillus, or Streptococcus.

The invention provides application of the promoter, the recombinant vector or the recombinant cell in expressing a target gene or an anabolic product.

The invention provides application of the promoter, the recombinant vector or the recombinant cell in preparation of protein or polypeptide drugs.

The invention provides a constitutive lactobacillus promoter, the function and the expression efficiency of the promoter are discovered and verified for the first time, and experiments show that the promoter can efficiently start the expression of downstream genes. With the conventional promoter P_NisinACompared with the prior art, the protein expression activity is obviously improved, and the protein is an ultra-efficient promoter. The promoter provided by the invention can be used for high-efficiency expression of endogenous or exogenous proteins of prokaryotes, and provides a favorable tool for development of protein medicine oral drugs taking probiotics as a delivery system.

Drawings

FIG. 1 is a graph comparing results of expression of human PD-1 ectodomain protein using different kinds of promoters, wherein FIG. 1-A is an electrophoretogram of expression of a gene encoding human PD-1 ectodomain protein using different kinds of promoters; FIG. 1-B is a bar graph showing the expression level of the gene encoding human PD-1 ectodomain protein using different types of promoters;

FIG. 2 is a graph comparing the results of the expression of fusion proteins of human PD-1 ectodomain and human TGF-beta R2 ectodomain using different promoters; wherein FIG. 2-A is an electrophoretogram of the fusion protein expressed using different promoters, and FIG. 2-B is a histogram of the expression level of the fusion protein expressed using different promoters;

in the above figures, denotes p < 0.05.

Detailed Description

The invention provides a constitutive lactobacillus promoter, wherein the nucleotide sequence of the promoter is shown as SEQ ID No.1 of a sequence table or is a complementary sequence shown as SEQ ID No.1 of the sequence table. The above sequence limitations of the promoter should not be understood as limitations of the promoter, but also include a nucleotide sequence having at least 70% homology with the nucleotide sequence shown as SEQ ID No. 1; the promoter comprises a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more nucleotides on the basis of the sequence shown as SEQ ID No. 1. The source of the promoter is not particularly limited in the present invention, and a promoter sequence synthesis method well known in the art may be used. In the examples of the present invention, the promoter was synthesized by Shanghai Bioengineering Co., Ltd.

The invention provides a recombinant vector containing the promoter.

In the present invention, the basic vector of the recombinant vector is not particularly limited, and a vector well known in the art may be used. In order to ensure the safety of subsequent application, the basic vector in the recombinant vector is preferably pNZ 8149; the pNZ8149 is a food grade plasmid; the recombinant vector uses the promoter to replace the original promoter P in pNZ8149_NisinA。

In the present invention, the recombinant vector further includes a target gene. The recombinant vector comprising the promoter of the present invention can be used for expressing any target gene. In order to better show that the promoter can efficiently express genes, the invention takes the expression sequence of an inhibitor for encoding a human PD-1/PD-L1 signal pathway and the expression sequence of an inhibitor for inhibiting a PD-1/PD-L1 signal pathway and a TGF beta/TGF beta R2 signal pathway as target genes.

In the invention, the target genes are sequences encoding inhibitors of the human PD-1/PD-L1 signaling pathway and sequences encoding inhibitors of the PD-1/PD-L1 signaling pathway and the TGF beta/TGF beta R2 signaling pathway. The sequence encoding an inhibitor of the human PD-1/PD-L1 signaling pathway preferably comprises a nucleotide sequence of a human PD-1 ectodomain structural protein or a sequence encoding a fusion protein comprising a human PD-1 ectodomain structural protein; the nucleotide sequence of the human PD-1 ectodomain structural protein is shown in SEQ ID No. 2; the sequence for encoding the fusion protein containing the human PD-1 ectodomain structural protein is a sequence for encoding human PD-1 ectodomain-human TGF beta R2 ectodomain fusion protein; the sequence encoding the fusion protein comprising the human PD-1 ectodomain structural protein is representative of an antibody/antibody fragment-factor fusion protein. The PD-1 ectodomain structural protein also comprises an analogue shown as SEQ ID No. 2; the analogue comprises a nucleotide sequence which has at least 70 percent of homology with the nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence which is obtained by modifying, substituting, deleting or adding one or more nucleotides on the basis of the sequence shown as SEQ ID No. 1. The sequence for coding the inhibitor for inhibiting the PD-1/PD-L1 signal pathway and the TGF beta/TGF beta R2 signal pathway is preferably a sequence for coding a human PD-1 ectodomain-human TGF beta R2 ectodomain fusion protein. The sequence of the fusion protein for encoding the human PD-1 ectodomain-human TGF beta R2 ectodomain is preferably shown as SEQ ID No. 3.

In the present invention, the method for preparing the recombinant vector is preferably a method in which a promoter (P)_gapA) The nucleotide sequence is inserted into restriction sites BstYI and NcoI of the plasmid to form a recombinant vector pGapa;

when a recombinant vector containing a target gene is prepared, it is preferable that the target gene is inserted between the protein expression vector pNZ8149, the pGapa restriction site SphI and the XbaI restriction site.

The present invention provides a recombinant cell comprising preferably said promoter or preferably said recombinant vector.

In the present invention, the basal cells in the recombinant cells preferably include intestinal normal bacteria; the intestinal normal bacteria preferably include Bifidobacterium, Lactobacillus, or Streptococcus.

In the present invention, the Bifidobacterium includes Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium infantis), and Bifidobacterium longum (Bifidobacterium longum).

The Lactobacillus (Lactobacillus) includes Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus crispatus (Lactobacillus crispus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus fermentum (Lactobacillus fermentium), Lactobacillus grisea (Lactobacillus gasseri), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus salivarius (Lactobacillus salivarius).

The Streptococcus (Streptococcus) includes Streptococcus thermophilus (Streptococcus thermophilus) and Staphylococcus calves (Staphylococcus vitulinus), Staphylococcus xylosus (Staphylococcus xylosus), Staphylococcus carnosus (Staphylococcus carnosus), Bacillus coagulans (Bacillus coagulons), Bacillus subtilis (Bacillus subtilis) and lactococcus lactis (Lactococcus lactis). The lactic acid bacterium is preferably L.1actasnz3900.

In the present invention, the method for preparing the recombinant cell comprises introducing the recombinant vector into a host cell by electrotransformation to obtain the recombinant cell. The method of the electric conversion is not particularly limited in the present invention, and a method of electric conversion well known in the art may be used.

The invention provides application of the promoter, the recombinant vector or the recombinant cell in expressing a target gene or an anabolic product.

The present invention does not impose any limitation on the kind of the target gene and the kind of the metabolite.

The invention provides application of the promoter, the recombinant vector or the recombinant cell in preparation of protein or polypeptide drugs.

In the present invention, the kind of the protein-based or polypeptide-based drug is not particularly limited. The diseases treated by the drug are not limited at all, and the types of the diseases treated by the drug comprise metabolic diseases, cancers, infectious diseases, immune diseases, polypeptide and protein drugs used for nervous system diseases; the metabolic disease is preferably selected from diabetes, obesity, diabetic kidney injury, diabetic macroangiopathy, diabetic microvascular injury, diabetic retinopathy, glucagon blood disease, necrotic wandering erythema, glucagonomas and other metabolic diseases, coronary heart disease, hypertensive heart disease, valvular heart disease, alcoholic cardiomyopathy or diabetic cardiovascular complications. The cancer is preferably lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, melanoma, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer. The infectious disease is preferably an HIV viral infection and a viral hepatitis viral infection, a rabies viral infection or varicella. The immune diseases are preferably autoimmune hepatitis, viral hepatitis, hepatic fibrosis, acute lung injury, asthma, inflammatory bowel disease, psoriasis, systemic lupus erythematosus or rheumatoid arthritis. The neurological disease is preferably epilepsy, stroke, Huntington's disease, Alzheimer's disease or Parkinson's disease.

In the invention, the preparation of the protein or polypeptide medicament takes probiotics as a platform, and the polypeptide or protein with a therapeutic effect is expressed by utilizing the recombinant vector carrying the promoter, so that the oral medicament delivery of the peptide or protein medicament is realized.

The preparation method of the drug delivery system comprises the following steps: the method comprises the following steps:

1) synthesizing a nucleotide sequence of a polypeptide or protein of interest;

2) inserting the nucleotide sequence synthesized in the step 1) into the vector containing the promoter to construct a recombinant expression vector;

3) transforming the recombinant expression vector in the step 2) into intestinal normal bacteria;

4) inoculating the recombinant bacteria obtained in the step 3) into a culture medium for culture and expression.

Alternatively, the method comprises the steps of:

1) synthesizing a promoter-nucleotide sequence of a polypeptide or protein of interest;

2) inserting and integrating the nucleotide sequence synthesized in the step 1) into a chromosome of the intestinal tract normal bacteria;

3) inoculating the recombinant bacteria obtained in the step 2) into a culture medium for culture and expression.

In the present invention, the pharmaceutical composition comprises a promoter-nucleotide sequence expression cassette for a polypeptide or protein of interest and a host cell. In the present invention, the pharmaceutical composition is preferably an oral pharmaceutical composition; the pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier and/or adjuvant. The dosage form of the medicament is preferably a capsule or other acceptable preparation.

The constitutive lactic acid bacteria promoter, the recombinant vector, the recombinant bacteria thereof and the application thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.