阅读说明：本技术 一种核糖体失活蛋白基因病毒载体构建及其在肿瘤细胞中表达活性蛋白的方法 (Construction of ribosome inactivating protein gene virus vector and method for expressing active protein in tumor cell ) 是由 刘梦铃 于 2019-10-29 设计创作，主要内容包括：本专利申请属于生物工程技术领域,具体公开了一种核糖体失活蛋白基因病毒载体构建及其在肿瘤细胞中表达活性蛋白的方法,包括如下步骤：步骤一、选取合适的核糖体失活蛋白,对其成熟区基因进行人源化表达的密码子优化；步骤二、野生型及优化后基因的腺病毒载体构建与重组病毒包装；步骤三、重组腺病毒感染肿瘤细胞,并检测使其在肿瘤细胞中蛋白表达及其抗肿瘤效应。本方案将α-苦瓜素基因进行密码子优化,构建能感染哺乳动物肿瘤细胞的腺病毒表达载体系统,以适合于在肿瘤细胞中表达活性蛋白,克服了现有技术中使用原核细胞表达体系只能在大肠杆菌中表达以及使用植物病毒载体只能在植物细胞中表达而不能在肿瘤细胞中表达和杀伤肿瘤的缺陷。(The application belongs to the technical field of biological engineering, and particularly discloses a ribosome inactivating protein gene virus vector construction and a method for expressing active protein in tumor cells, which comprises the following steps of selecting proper ribosome inactivating protein, carrying out codon optimization of humanized expression on mature region genes, constructing wild adenovirus vectors and optimized genes, packaging recombinant viruses, infecting tumor cells by the recombinant adenovirus, and detecting protein expression and anti-tumor effect in the tumor cells.)

1. A ribosome inactivating protein gene virus vector construction and a method for expressing active protein in tumor cells are characterized by comprising the following steps:

selecting proper ribosome inactivating protein, and optimizing humanized codons of the ribosome inactivating protein;

cloning Wild α -MMC gene, namely using plasmid as a template to obtain Wild Type-Type sequence, designing a primer to perform PCR reaction, cloning the gene into a pDC316-mCMV-EGFP adenovirus shuttle vector after enzyme cutting, and performing sequencing verification, (2) performing complete sequence synthesis on the sequence after codon optimization in the step one, (3) respectively inserting the Wild Type and the optimized α -MMC gene into the pDC316-mCMV-EGFP adenovirus shuttle vector, and performing sequencing verification, (4) respectively cotransfecting AdMax293 cells with the shuttle vector and adenovirus skeleton plasmid pBHGlox _ E1 and 3Cre, and performing homologous recombination to obtain the Wild Type recombinant adenovirus and the optimized recombinant adenovirus containing target gene;

and step three, transfecting the recombinant adenovirus obtained in the step two to tumor cells, expressing active protein in the tumor cells, and detecting the anti-tumor effect.

2. The method for constructing a ribosome inactivating protein gene viral vector according to claim 1 and expressing active protein in tumor cells thereof, wherein the ribosome inactivating protein selected in the step one has RNA N-glycosidase activity, has highly conserved active cleft residues and secondary structures in the active site region, and acts on eukaryotic cell large subunit 28S to inactivate ribosomes and inhibit the biosynthesis of the protein.

3. The method according to claim 2, wherein the ribosome inactivating protein gene selected in the step one is selected from any one of the group consisting of type I ribosome inactivating proteins such as α -charantin, MAP30, β -charantin, γ -charantin, δ -charantin, trichosanthin, pokeweed protein, luffa and saporin, and type II ribosome inactivating proteins such as ricin A chain.

4. The method for constructing a ribosome inactivating protein gene viral vector according to claim 3 and expressing active protein in tumor cells, wherein the gene of ribosome inactivating protein is α -momordicin gene.

5. The method of claim 4, wherein said step A comprises using three specialized codon Optimization software to simultaneously optimize α momordicin mature region, including MaxCodon Optimization Program, DNAworks, Synthetic Gene Designer, and combining the advantages of each software to obtain ideal Optimization results.

6. The method of claim 5, wherein the adenovirus vector in step two is a mammalian cell expression system adenovirus vector, or a ribosome inactivating protein vector constructed from any one of adenovirus, retrovirus, and lentivirus.

The technical field is as follows:

the invention belongs to the technical field of bioengineering, and particularly relates to a ribosome inactivating protein gene virus vector construction method and a method for expressing active protein in tumor cells.

Background art:

ribosome Inactivating Proteins (RIPs) are toxic Proteins mainly distributed in plants and have RNA N-glycosidase activity. RIPs have highly conserved active cleft residues and secondary structures in an active site region, and can act on the eukaryotic cell large subunit 28S to inactivate ribosome and inhibit protein biosynthesis, thereby exerting a cytotoxic effect on cells. RIPs are divided into three types, type I RIP is single peptide chain protein with the molecular weight of about 1 l-30 kDa, such as balsam pear ribosome inactivating protein, Trichosanthin (TCS), pokeweed protein (PAP), luffa poison protein (1uffin), saporin and the like, and most ribosome inactivating proteins belong to type I; type II RIP is a dimeric protein, such as ricin, which has not only a toxic polypeptide A chain that inactivates ribosomes, but also a binding polypeptide B chain; type III is less common. RIPs have pharmacological activities of reducing blood sugar, resisting fertility, resisting tumor and virus, treating AIDS and the like, and have wide clinical application prospect.

At present, Lee-Huang et al utilize pRSET expression vector to express MAP30 gene in Escherichia coli, find that non-glycosylated recombinant MAP30 has the same activity as natural MAP30, Arazi T et al (2002) report that recombinant MAP30 protein produced by cucurbitaceae special-purpose virus vector pumpkin yellow mosaic virus (ZYMV-AG1I) vector has antiviral and antitumor activities, national scholars Zhongpeng (2005), Huangqianming (2007), Zhanjinbiao (2010) and the like successfully construct α -MMC and MAP30 expression systems in sequence, and obtain bioactive balsam pear ribosome inactivating protein.

However, neither the ribosome-inactivating protein is expressed in E.coli by using a prokaryotic vector or expressed in a plant virus vector system, nor the ribosome-inactivating protein is expressed in mammalian cells, and thus, it cannot be used for anti-tumor gene therapy.

The invention content is as follows:

in view of the above-mentioned disadvantages, the present invention aims to provide a construction of a ribosome inactivating protein gene viral vector and a method for expressing an active protein in a tumor cell.

In order to achieve the purpose, the basic scheme of the invention is as follows:

a method for constructing a ribosome inactivating protein gene virus vector and expressing active protein in tumor cells by the ribosome inactivating protein gene virus vector comprises the following steps:

selecting proper ribosome inactivating protein, and optimizing humanized codons of the ribosome inactivating protein;

cloning Wild α -MMC gene, namely using plasmid as a template to obtain Wild Type-Type sequence, designing a primer to perform PCR reaction, cloning the gene into a pDC316-mCMV-EGFP adenovirus shuttle vector after enzyme cutting, and performing sequencing verification, (2) performing complete sequence synthesis on the sequence after codon optimization in the step one, (3) respectively inserting the Wild Type and the optimized α -MMC gene into the pDC316-mCMV-EGFP adenovirus shuttle vector, and performing sequencing verification, (4) respectively cotransfecting AdMax293 cells with the shuttle vector and adenovirus skeleton plasmid pBHGlox _ E1 and 3Cre, and performing homologous recombination to obtain the Wild Type recombinant adenovirus and the optimized recombinant adenovirus containing target gene;

and step three, transfecting the recombinant adenovirus obtained in the step two to tumor cells, expressing active protein in the tumor cells, and detecting the anti-tumor effect.

Furthermore, the ribosome inactivating protein obtained in the step one has RNA N-glycosidase activity, namely has highly conserved active cleft residues and secondary structures in an active site region, can act on eukaryotic cell large subunit 28S to inactivate ribosomes, inhibit the biosynthesis of protein, and can generate cytotoxic action on tumor cells to kill tumors.

Further, the ribosome inactivating protein gene may be any one of α -charantin, MAP30, β -charantin, gamma-charantin, delta-charantin, trichosanthin, pokeweed protein, luffa toxin protein, saporin and other type I ribosome inactivating protein and type II ribosome inactivating protein (such as ricin A chain).

Further, the gene of the ribosome inactivating protein is α -charantin gene, including wild type and optimized α -charantin gene.

Furthermore, in the step A, three professional codon Optimization software are used for simultaneously carrying out mammal codon Optimization on the α charantin mature region, namely MaxCodon Optimization Program, DNAworks and synthetic Gene Designer, and the advantages of the software are integrated to obtain an ideal Optimization result, so that the expression quantity and the expression efficiency of the target gene in the mammal cell and the tumor cell are improved.

Further, the adenovirus vector in the second step is a ribosome inactivating protein carrier constructed by any one of adenovirus vectors of a mammalian cell expression system and adenovirus vectors, retrovirus vectors, lentivirus vectors and the like; the scheme uses the adenovirus vector of a mammalian cell expression system, and other vector systems also have the same vector function, so that the ribosome inactivating protein carrier constructed by virus vectors such as adenovirus, adeno-associated virus, retrovirus, lentivirus and the like is also suitable.

The technical effects are as follows:

the proposal takes α -charantin gene as an experimental model to construct a gene expression virus recombinant containing ribosome inactivating protein, wherein the ribosome inactivating protein has RNA N-glycosidase activity and can inactivate the ribosome of tumor cells and inhibit protein synthesis to generate cytotoxic action on the tumor cells.

Compared with the prior art, the method can ensure that the expression quantity and sales of the target protein are higher; the expressed toxicological effect is more obvious, the two recombinant viruses have obvious cytotoxic effects on breast cancer MCF-7 cells and cervical cancer Hela cells, and the optimized effect is more obvious; the two recombinant viruses have obvious inhibition effect on the growth of breast cancer MCF-7 cells and cervical cancer Hela cells.

The ribosome inactivating protein in the scheme has RNA N-glycosidase activity, so the recombinant virus containing the ribosome inactivating protein gene is also suitable for cells of breast cancer, liver cancer, lung cancer, gastric cancer, colon cancer, bladder cancer, choriocarcinoma, cervical cancer, trophoblastic carcinoma and melanoma.

Description of the drawings:

FIG. 1 is a schematic flow chart of the present invention

FIG. 2 is a schematic diagram of analysis of amino acid expression rate before codon optimization of α charantin gene of the present invention;

FIG. 3 is a schematic diagram of analysis of codon preference and amino acid expression rate of α charantin gene after codon optimization;

FIG. 4 adenovirus shuttle vector map;

FIG. 5 is a schematic diagram of optimized sequence shuttle plasmid vector pDC316-MMC-mCMV-EGFP enzyme digestion;

FIG. 6 is a schematic representation of the sequencing validation of the optimized sequence shuttle plasmid vector α Momordicaxin gene;

FIG. 7 is a schematic diagram of the PCR results of the wild type sequence shuttle plasmid vector pDC316-MMCwt-mCMV-EGFP construction;

FIG. 8 is a schematic diagram of the double digestion of pDC316-MMCwt-mCMV-EGFP adenovirus vector and PCR-recovered product;

FIG. 9 is a schematic alignment of the sequencing results of wild type sequence shuttle plasmid vector pDC 316-MMCwt-mCMV-EGFP;

FIG. 10 is a schematic diagram of a two-step QPCR process;

FIG. 11 is a schematic representation of the dissolution curves for wild-type momordicin and the optimized codon momordicin;

FIG. 12 is a fluorescence image of 293T cell transfection;

FIG. 13 is a diagram showing WB detection results;

FIG. 14 is a schematic diagram showing the results of infection of breast cancer MCF-7 and cervical cancer Hela with three viruses, i.e., unloaded adenovirus Adv-mCMV, recombinant adenovirus Adv-MMCwt and recombinant adenovirus Adv-MMC;

FIG. 15 is a graph showing an absorbance value (OD value) curve of MTT-method-detecting cells;

FIG. 16 is a schematic diagram showing the results of cytopathic effect observed 5 days after the recombinant adenovirus infects tumor cells;

FIG. 17 is a schematic diagram showing the effect of inhibiting tumor cell growth by recombinant virus infection.

Brief description of the sequence listing (I) shows the nucleotide sequence of α charantin gene after codon optimization

Detailed Description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the motion situation, etc. in a specific posture, if the specific posture is changed, the directional indicator is changed accordingly.

In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.