阅读说明：本技术 一种HTLV-1 Env介导的细胞-细胞融合模型、制备方法及应用 (HTLV-1 Env mediated cell-cell fusion model, preparation method and application ) 是由 段良伟 王辉 郑倩倩 张赟 牛玉娜 张阳 于 2020-07-09 设计创作，主要内容包括：本发明属于生物医学技术领域,尤其涉及一种HTLV-1 Env介导的细胞-细胞融合模型、制备方法及应用。本申请中获得HTLV-1 Env基因片段,并替换载体pSRHS-Tat-Env<Sub>HIV-1</Sub>的HIV-1 Env基因中对应的gp145片段,转染效应细胞COS-1,并与基因组中整合有LTR及其下游串联着的萤火虫荧光素酶报告基因的TZM-bl细胞共培养；通过检测萤火虫荧光素酶的活性间接检测HTLV-1 Env介导的细胞-细胞融合能力。利用抑制多肽P-400的对该融合系统进行验证,表明该系统非常适用于针对HTLV-1病毒进入抑制剂的筛选和验证,以及对进入抑制剂有效浓度和毒性等指标进行评估。(The invention belongs to the technical field of biomedicine, and particularly relates to an HTLV-1 Env mediated cell-cell fusion model, a preparation method and application. In the application, an HTLV-1 Env gene fragment is obtained and replaces a vector pSRHS-Tat-Env HIV‑1 Transfecting an effector cell COS-1 with a gp145 fragment corresponding to the HIV-1 Env gene, and co-culturing with TZM-bl cells integrated with LTR and a firefly luciferase reporter gene connected downstream of the LTR in series in the genome; HTLV-1 Env-mediated cell-cell fusion capacity was indirectly measured by measuring the activity of firefly luciferase.The fusion system is verified by utilizing the inhibitory polypeptide P-400, which shows that the fusion system is very suitable for screening and verifying the entry inhibitor of the HTLV-1 virus and evaluating the indexes such as effective concentration, toxicity and the like of the entry inhibitor.)

1. Recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env_HTLV-1The method for constructing (1), characterized in that,the method comprises the following steps:

plasmid pSRHS-Tat-Env_HIV-1CRF07Mutating the first amino acid residue behind the corresponding gp145 fragment in HIV-1 Env into a stop codon to obtain a mutant; replacing gp145 fragment of HIV-1 Env gene of the mutant with HTLV-1 Env full-length fragment to obtain recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env_HTLV-1。

2. The recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env according to claim 1_HTLV-1The construction method of (2), characterized in that: and mutating the amino acid residue into a stop codon by using a rolling circle PCR technology, wherein the sequence of a point mutation primer related to the rolling circle PCR technology is shown in SEQ ID NO.2 and SEQ ID NO. 3.

3. A recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env according to claim 2_HTLV-1The construction method of (2), characterized in that: rolling circle PCR method point mutation reaction system:

the rolling circle PCR method point mutation reaction conditions are as follows:

4. the recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env according to claim 1_HTLV-1The construction method of (2), characterized in that: the sequence of the HTLV-1 Env fragment is shown in SEQ ID NO. 4.

5. The recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env according to claim 1_HTLV-1The construction method of (2), characterized in that: obtaining a recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env_HTLV-1Then transformed into Top10 competent cells.

6. A recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env according to any one of claims 1 to 5_HTLV-1The plasmid pSRHS-Tat-Env constructed by the construction method of (1)_HTLV-1。

7. The plasmid pSRHS-Tat-Env of claim 6_HTLV-1The application of the protein in an HTLV-1 Env mediated cell-cell fusion system.

8. The plasmid pSRHS-Tat-Env of claim 6_HTLV-1Use in screening and/or validation of entry inhibitors against HTLV-1 virus.

9. An HTLV-1 Env-mediated cell-cell fusion system comprising: effector COS-1, plasmid pSRHS-Tat-Env as defined in claim 6_HTLV-1And target cells TZM-bl.

10. Use of an HTLV-1 Env mediated cell-cell fusion system according to claim 9 for screening and/or validation of entry inhibitors against HTLV-1 virus.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an HTLV-1 Env mediated cell-cell fusion model, a preparation method and application.

Background

Human T-lymphoblastic leukemia type 1 virus (HTLV-1), the first retrovirus demonstrated to be directly associated with human cancer as reported in 1980 by Robert-Gello research group, national cancer institute. Currently, the number of infected persons worldwide is more than ten million, and the infection can induce various diseases such as adult T cell leukemia (ATL) and HTLV-1 related myelopathy (HAM)/tropical spastic paraparesis (also called as spastic paraparesis, TSP). Despite the research of 40 years, until now there is no specific therapy and safe and long lasting effective vaccine formulation against HTLV-1 infection, which is an endemic disease that seriously threatens human health and puts a great pressure on the development of the global socioeconomic sector.

The entry inhibitor is an important anti-enveloped virus drug, blocks a central link of virus replication, namely invasion of target cells at the early stage of virus infection, can prevent viruses from infecting new host cells, reduces the virus load in blood plasma, has obvious superiority, is more and more concerned and emphasized by scientific researchers and drug developers, and has the potential of becoming an effective therapy for HTLV-1 virus infection. The screening and identification of new potent entry inhibitors are of great significance to human health and social development.

Viruses are microorganisms with extremely simple structures, do not have complete metabolic enzyme systems, and need to synthesize substances by host cells so as to complete the life cycle of the viruses. HTLV-1 is also an enveloped virus (also known as an enveloped virus) that first invades the host cell via membrane fusion to initiate a new life cycle and is therefore the primary link in the entire viral replication cycle. Enveloped viruses generally enter host cells by two means. In the first, envelope glycoproteins located on the surface of the virus bind to the receptor, inducing conformational changes that result in direct fusion of the viral envelope and host cell membrane at the cell surface. In the second, the virus invades the cell via an endocytic pathway, localizes to the endosomes, triggers a conformational change in the envelope glycoprotein in a pH and/or protease dependent manner, and thus triggers membrane fusion.

It has been found by researchers for a long time that, unlike human immunodeficiency virus type 1 (HIV-1), free HTLV-1 virions are paired with CD4⁺The infection efficiency of T lymphocytes is quite low, which applies to most HTLV-1 target cell types other than dendritic cells. Recent studies have found that free HTLV-1 virions can efficiently infect dendritic cells. Meanwhile, infected dendritic cells can rapidly and repeatedly transfer HTLV-1 to autologous CD4⁺T cells, causing CD4⁺Long-term prolific infection of T cells and results in interleukin-2-independent transformation of T cells. Dendritic cells, which are important antigen presenting cells, are considered to be the first cells encountered after invasion by various pathogens, and thus are likely to play a key role in the transmission of HTLV-1. Alais et al demonstrated by elaborate experiments that dendritic cells are more sensitive to infection by HTLV-1 than autologous lymphocytes and can efficiently transfer the virus to lymphocytes. Thus, dendritic cells, which are the first cells to be infected with HTLV-1 in vivo, are likely to represent a true viral pool.

Invasion of target cells such as dendritic cells by free HTLV-1 virus is mediated by the viral envelope glycoprotein envelope (env). HTLV-1 Env belongs to a type I transmembrane protein, is synthesized in a 488-amino acid precursor protein form in a rough endoplasmic reticulum, is folded and accompanied by oligomerization and initial glycosylation, is then transported to a Golgi apparatus, is cut by furin-like protease family to form mature surface glycoprotein gp46 and transmembrane glycoprotein gp21, and gp46 and gp21 are still non-covalently linked together to form heterodimers after being cut, and finally forms a trimeric spike consisting of three gp46 molecules and three gp21 molecules. Gp46 contains a receptor binding domain, which determines viral tropism and initiates viral infection; gp21 contains a trimerization domain, which dominates the trimerization of Env, and a membrane fusion domain, which mediates the membrane fusion process of virus and cells, completing virus invasion. Correspondingly, Env trimers located on the surface of target cells mediate fusion between target cells and receptor-expressing cells to form multicellular bodies. Furthermore, numerous studies have shown that Env is important for the formation of viral synapses of HTLV-1, and therefore Env plays a very important role in the cell-cell spread of HTLV-1 as well, and is associated with the ability to induce syncytial formation.

HTLV-1 Env plays a crucial role in the replication cycle, cell tropism and pathogenicity of the virus and is thus the most important and promising potential vaccine component and important antiviral drug target. An entry inhibitor against HTLV-1 Env can inhibit the entry of HTLV-1 virus into target cells, thereby inhibiting the spread of the virus in the initial stage of infection, and thus is one of the best ways to treat and prevent HTLV-1 infection at an early stage. The high-efficiency accurate invasion inhibitor screening model is necessary guarantee for obtaining medicines, at present, a plurality of cell-cell fusion models related to HIV-1 exist, and for HTLV-1, because the concerned degree is far less than that of HIV-1, the existing HTLV-1 envelope glycoprotein Env-mediated cell-cell fusion models have no high efficiency, convenience and quickness of HIV-1.

Disclosure of Invention

The invention provides an HTLV-1 Env mediated cell-cell fusion model, a preparation method and application aiming at solving part of problems in the prior art or at least alleviating part of problems in the prior art.

The invention is realized in such a way that a recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env_HTLV-1The construction method comprises the following steps:

plasmid pSRHS-Tat-Env_HIV-1CRF07Mutating the first amino acid residue (G713, according to the code of international standard strain HXB 2) behind the corresponding gp145 fragment in the HIV-1 Env into a stop codon to obtain a mutant;

replacement of the mutant with a full-length fragment of HTLV-1 EnvObtaining a gp145 fragment of the HIV-1 Env gene to obtain a recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env_HTLV-1。

After mutation is a stop codon, the protein expressed by the mutant is gp145, and the sequence is shown in SEQ ID NO. 1. Thereafter, the gp 145-expressing fragment of the mutant was removed, and the HTLV-1 Env-expressing fragment was ligated to a linearized vector from which the gp145 fragment was removed by inverse PCR, replacing the gp145 portion of the HIV-1 Env gene.

Furthermore, the G713 amino acid residue is mutated into a stop codon by using a rolling circle PCR technology, and the sequence of a point mutation primer related to the rolling circle PCR technology is shown in SEQ ID NO.2 and SEQ ID NO. 3.

Further, a linearized vector for removing the gp145 segment is obtained by using an inverse PCR technology, and primer sequences related to the inverse PCR technology are shown in SEQ ID NO.7 and SEQ ID NO. 8.

Further, a rolling circle PCR method point mutation reaction system:

the rolling circle PCR method point mutation reaction conditions are as follows:

further, the sequence of the HTLV-1 Env fragment is shown in SEQ ID NO. 4.

Further, a recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env was obtained_HTLV-1Then transformed into Top10 competent cells.

The recombinant HTLV-1 Env expression plasmid pSRHS-Tat-Env_HTLV-1The plasmid pSRHS-Tat-Env constructed by the construction method of (1)_HTLV-1。

Plasmid pSRHS-Tat-Env as described above_HTLV-1The application of the protein in an HTLV-1 Env mediated cell-cell fusion system.

Plasmid pSRHS-Tat-Env as described above_HTLV-1Use in screening and/or validation of entry inhibitors against HTLV-1 virusThe application is as follows.

An HTLV-1 Env mediated cell-cell fusion system comprising: effector COS-1, plasmid pSRHS-Tat-Env as defined in claim 6_HTLV-1And target cells TZM-bl.

Use of an HTLV-1 Env mediated cell-cell fusion system as described above for screening and/or validation of entry inhibitors against HTLV-1 virus.

The invention applies a cell-cell fusion model of mature HIV-1 to HTLV-1 virus. By transfection of pSRHS-Tat-Env_HIV-1Cells in which the plasmid co-expresses the HIV-1 envelope glycoprotein Env and the trans regulatory protein Tat are called effector cells (i.e., COS-1 cells in this application). The HIV-1 indicator cell line TZM-bl is selected as a target cell, is actually a derivative cell strain of HeLa, stably and highly expresses a CD4 receptor and CCR5 and CXCR4 coreceptors on a cell membrane, and integrates HIV-1 long terminal repeat sequence LTR containing a Tat response element in a genome, so as to start the expression of a firefly luciferase (firefly luciferase) reporter gene which is connected in series downstream of the LTR. When the effector cell and the target cell are fused, the transactivator Tat in the effector cell can activate LTR in the genome of the target cell so as to start the high-efficiency expression of luciferase (Luc). Therefore, the detected luciferase activity can indirectly reflect the HIV-1 Env mediated intercellular fusion level.

Given that the HeLa cell line itself expresses multiple functional receptors required for HTLV-1 virus invasion, there is theoretically the possibility of applying the mature HIV-1 cell-cell fusion model to HTLV-1 virus. However, the intracellular domain of HIV-1 Env contains the second codon region of the Tat protein (encoding a 67-101 fragment of Tat), which, when replaced, may affect Tat function. In the present application, pSRHS-Tat-Env was first subjected to the rolling circle PCR method_HIV-1CRF07The G713 amino acid residue of HIV-1 Env (encoded by International Standard strain HXB 2) in the plasmid was mutated to a stop codon and the results showed that the ability of the HIV-1 envelope glycoprotein gp145 produced by the mutation to mediate cell-cell fusion was not affected and was even slightly elevated compared to the full-length protein. Thus, the present application can creatively use HTLV-1 Env to transform pSRHS-Tat-Env_HIV-1CRF07The gp145 partial replacement of HIV-1 Env on the plasmid achieved expression of the more toxic HTLV-1 envelope glycoprotein in effector cells, and the HIV-1Tat protein was unaffected. Compared with a control vector, the target plasmid can obtain a higher-level luciferase activation multiple, which indicates that HTLV-1 Env successfully mediates fusion between effector cells and target cells, the fusion capability of the HTLV-1 Env is almost the same as that of HIV-1 Env, and the HTLV-1 cell-cell fusion system has the advantages of rapidness, simplicity, convenience, high efficiency, strong sensitivity, good applicability and the like of the original HIV-1 cell-cell fusion system.

The model is further used for verifying the reported inhibition effect of the polypeptide P-400 corresponding to the HTLV-1 Env 400-429 amino acid, the activity of luciferase is reduced by more than 20 times at the concentration of 20 mu g/ml, the inhibition effect is better than reported data, and the model is particularly suitable for screening and verifying the entry inhibitor of HTLV-1 virus and evaluating indexes such as effective concentration and toxicity of the entry inhibitor.

In summary, the advantages and positive effects of the invention are:

firstly, pSRHS-Tat-Env is subjected to rolling circle PCR (polymerase chain reaction) method_HIV-1CRF07The 713 th amino acid (the first amino acid after the transmembrane region) of the HIV-1 Env in the plasmid is mutated into a stop codon, and the capability of the generated HIV-1 envelope glycoprotein gp145 form for mediating cell-cell fusion is not influenced at all and is even increased compared with the full-length protein. Then obtaining an HTLV-1 Env gene fragment by utilizing a polymerase chain reaction PCR technology, and using the HTLV-1 Env gene fragment to express a classical envelope expression vector pSRHS-Tat-Env_HIV-1CRF07The corresponding gp145 fragment in the HIV-1 Env gene is replaced, and effector cell COS-1 is transfected, so that HTLV-1 envelope protein Env and trans regulatory protein Tat are co-expressed. TZM-bl is selected as a target cell, the cell expresses HTLV-1 receptor, HIV-1 long terminal repeat LTR is integrated in the genome, and the expression of a firefly luciferase (firefly luciferase) reporter gene connected in series downstream is controlled. Because the fusion level of the effector cells and the target cells is positively correlated with the expression level of luciferase (Luc), HTLV-1 Env-mediated cell fusion capacity can be indirectly detected by detecting the activity of Luc, and the cell fusion capacity can be utilizedThe reported inhibition polypeptide P-400 verifies the fusion system, and the obtained inhibition effect is more than 20 times and is superior to the reported data. Therefore, the cell-cell fusion system has the advantages of rapidness, simplicity, convenience, high efficiency, strong sensitivity, good applicability and the like, and is very suitable for screening the entry inhibitor aiming at the HTLV-1 virus, verifying the entry inhibitor screened by other systems, and evaluating the indexes such as the effective concentration and toxicity of the entry inhibitor.

Drawings

FIG. 1 is a representation of gp 145-mediated cell-cell fusion with premature termination of HIV-1 Env formation;

FIG. 2 is a schematic diagram of the principle of HTLV-1 Env mediated cell-cell fusion system;

FIG. 3 is an HTLV-1 Env-mediated fusion of cells with target cells in effector cells;

FIG. 4 is a graph of the inhibitory effect of the entry inhibitor P-400 based on the HTLV-1 Env-mediated cell-cell fusion system.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

The genes, proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

The invention discloses an HTLV-1 Env mediated cell-cell fusion model, a preparation method and application, which are shown in the following embodiments.