WO2013091130A1 - 猪cd28受体，编码其的基因及其应用 - Google Patents

猪cd28受体，编码其的基因及其应用 Download PDF

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WO2013091130A1
WO2013091130A1 PCT/CN2011/002123 CN2011002123W WO2013091130A1 WO 2013091130 A1 WO2013091130 A1 WO 2013091130A1 CN 2011002123 W CN2011002123 W CN 2011002123W WO 2013091130 A1 WO2013091130 A1 WO 2013091130A1
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porcine
gene
receptor
cells
seq
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PCT/CN2011/002123
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English (en)
French (fr)
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索勋
刘贤勇
苏华荔
赵新新
黄骁舾
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中国农业大学
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Priority to US14/366,811 priority Critical patent/US9422360B2/en
Priority to PCT/CN2011/002123 priority patent/WO2013091130A1/zh
Publication of WO2013091130A1 publication Critical patent/WO2013091130A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag

Definitions

  • Porcine CD28 receptor gene encoding the same and its application
  • the invention belongs to the field of animal genetic engineering, and specifically relates to a porcine CD28 receptor, a gene encoding the same and application thereof. Background technique
  • the adaptive immune response plays a crucial role in the animal's resistance to pathogen invasion.
  • the activation of T cells is the most important of the reaction.
  • Recent studies have demonstrated that effective activation of T cells requires dual signal stimulation.
  • One is the first signal produced by the binding of MHC-antigen peptide to TCR, and the other is the co-stimulatory receptor, mainly the second signal produced by the binding of CD28 and its ligand.
  • the co-stimulatory receptor mainly the second signal produced by the binding of CD28 and its ligand.
  • antigen-presenting cells often present a small number of antigenic peptides, and their binding to TCR is not sufficient to activate T cells, which may easily cause T cell incompetence.
  • the T-stimulation receptor CD28 and other co-stimulatory signaling pathways can compensate for the weak TCR signal and activate T cells.
  • CD28 receptor is a typical representative of costimulatory receptors, and its gene sequence information and protein function are A detailed description of human and mouse studies has been made.
  • the CD28 receptor is a recognized co-stimulatory molecule for the initiation of proliferation and survival of T cells.
  • the receptor is linked to its ligand B7.1 (also known as CD80) or B7.2 (also known as CD86), which has a significant effect on the activation, proliferation and survival of T cells, and can affect the differentiation direction of T cells. And upregulate the expression of cytokines such as IFN- ⁇ . It has been found that the expression level of CD28 on the surface of T cells in the elderly is significantly down-regulated, and some cases with delayed activation of the immune system also show lower expression levels of CD28 molecules. Therefore, the expression level of CD28 molecule directly affects the effective activation of the immune system, thereby affecting the disease resistance of the individual.
  • CD28 receptors play an important role in the establishment, enhancement and maintenance of T cell immune responses, which indicates that they have good prospects in the research and application of transgenic breeding as target genes.
  • most of the current co-stimulatory molecular related studies are based on gene knockout or the use of anti-CD28 monoclonal antibodies to remove or enhance the costimulatory signal. These strategies are clearly not suitable for breeding research.
  • only the predicted gene sequence information of pig CD28 receptors has been published, and the exact coding sequence and its protein function have not been reported before. Summary of the invention
  • the present invention first provides a porcine CD28 receptor which is a protein consisting of the amino acid sequence shown by SEQ ID NO.
  • the porcine CD28 receptor of the present invention further comprises a protein represented by SEQ ID NO. 2 which is substituted, deleted or added with one or several amino acids and has an equivalent activity in the amino acid sequence shown by SEQ ID NO. protein.
  • SEQ ID NO. 2 a protein represented by SEQ ID NO. 2 which is substituted, deleted or added with one or several amino acids and has an equivalent activity in the amino acid sequence shown by SEQ ID NO. protein.
  • SEQ ID NO. 2 which is substituted, deleted or added with one or several amino acids and has an equivalent activity in the amino acid sequence shown by SEQ ID NO. protein.
  • the serine at position 181 is replaced with threonine, or the glutamine at position 188 is deleted, or three prolines are added after position 198.
  • the amino acid sequence of the derivative protein may have a homology with the amino acid sequence of SEQ ID No. 2 of 70% or more, preferably 80% or more, more preferably 90% or more.
  • the invention also provides a gene encoding the above protein.
  • the nucleotide sequence of the gene encoding the porcine CD28 receptor provided by the present invention is shown in SEQ ID No. 1.
  • the vectors, cell lines and host bacteria containing the porcine CD28 receptor gene also provided are within the scope of the present invention.
  • the invention also provides the use of the porcine CD28 receptor for increasing broad-spectrum disease resistance in pigs.
  • the binding of the monoclonal antibody to the CD28 receptor can enhance the second signal required for T cell activation, thereby enhancing the T cell immune response.
  • the present invention also provides the use of the above porcine CD28 receptor gene for cultivating a broad spectrum of disease resistant pigs.
  • the specific method is:
  • the transgenic strategy based on the costimulatory receptor CD28 of the present invention is a novel strategy for animal breeding research.
  • the co-stimulatory receptor CD28 is specifically expressed in T cells, which can enhance the activation, proliferation and cytokine secretion activity of T cells when stimulated by the antigen, thereby enhancing the host's acquired immune response and enhancing the vaccine immune effect. Therefore, those skilled in the art can foresee that after the successful cultivation of the transgenic animal, the immune function and disease resistance of the pathogen infection will also be significantly improved.
  • the host Based on the strategy of high expression of CD28, compared with the new breed of livestock that cultivates a single disease resistance characteristic, the host can more effectively enhance the disease resistance of the host when cross-infected with multiple pathogens, and improve the vaccine effect.
  • FIG. 1 is a schematic diagram of the vector pIRES-CD28HA, in which CMV: cytomegalovirus (CMV) promoter, a promoter that regulates expression of a gene of interest in eukaryotic cells; CD28: porcine CD28 gene coding region; HA: encodes an HA (Influenza virus hemagglutinin antigenic determinant) A gene sequence of a short peptide that can be used as a tag to detect the expression of the fusion protein; IRES: The 5' end of eukaryotic mRNA has a short RNA sequence that can be independently initiated EGFP: an enhanced green fluorescent protein gene coding region; SV40: is a polyadenylation residue at the end of the monkey vacuolar virus 40 mRNA, which has the effect of terminating transcription and enhancing the stability of RNA.
  • CMV cytomegalovirus
  • CD28 porcine CD28 gene coding region
  • HA encodes an HA (Influenza virus hemag
  • Figure 2 shows the high expression of CD28 by flow cytometry after transfection of murine T cells with murine CD28 mRNA.
  • transfected 2 ⁇ g CD28 mRNA, the number of T cells with high expression of CD28 increased
  • B is the enhanced fluorescence intensity of T cells with high expression of CD28, that is, the number of CD28 molecules per surface of each T cell increased.
  • Figure 3 shows the up-regulation of the expression level of the cell surface activation marker (Marker) upon stimulation of the antigen-presenting system by transfecting murine T cells with murine CD28 mRNA.
  • A was transfected with 20 g CD28 mRNA, and the number of T cells with high expression of marker (CD25, CD44 and CD69) was increased.
  • B was enhanced by the high expression of Marker (CD25, CD44 and CD69). The number of Marker activated on each T cell surface increased.
  • Figure 4 shows that after transfected murine T cells with murine CD28 mRNA, the amount of IFN- ⁇ secreted by the cells was increased when stimulated by the antigen-presenting system, but the amount of IL-4 secreted was not significantly changed compared with cells that were not transfected with CD28 mRNA. .
  • Figure 5 shows that the differentiation direction of cells was significantly affected by transfection of murine T cells with murine CD28 mRNA.
  • Up-regulation of the transcription factors T-bet, GATA3 and RORyt indicated that more T cells differentiated into Thl, Th2 and TM7, which have positive immunoregulatory effects.
  • Down-regulation of Foxp3 factors indicates T cells in the direction of Treg (negative regulation) The differentiation is inhibited.
  • Figure 6 shows the expression of foreign proteins in pig peripheral blood mononuclear cells (PBMC) transfected with pIRES-CD28HA plasmid by Western blot.
  • PBMC peripheral blood mononuclear cells
  • Figure 7 shows that the transcription level of the T cell activation marker CD25 molecule is significantly increased when porcine peripheral blood mononuclear cells (PBMC) are transfected with pIRES-CD28HA plasmid and stimulated by antigen (PRRSV).
  • PBMC peripheral blood mononuclear cells
  • PRRSV antigen
  • Figure 8 shows that porcine peripheral blood mononuclear cells (PBMC) are significantly stimulated to increase the level of IFN- ⁇ in PBMC cells when stimulated with antigen (PRRSV) after transfection with pIRES-CD28HA plasmid.
  • the invention adopts the high similarity between the human and pig gene sequence alignment results as a theoretical basis, and designes the primer according to the human CD28 sequence, and uses the cDNA of Wuzhishan pig as a template to retrieve the suspected CD28 sequence of the pig.
  • the specific steps are as follows: (1) By comparing with the human CD28 gene sequence, a gene fragment with higher similarity was found in the pig genome, and primers were designed accordingly (see Table 1: P1 and P2).
  • RNA of PBMC was extracted using Invitrogen's Trizol reagent (TRIzol® LS Reagent), and porcine cDNA was synthesized using ABI's First Strand Synthesis Kit for RT-PCR.
  • Amplification reaction system double distilled water, 34.5 ⁇ l; 5 x HF buffer, ⁇ ; lOmM dNTP mix, ⁇ ; 25 ⁇ ⁇ 1, ⁇ ; 25 ⁇ ⁇ 2, ⁇ ; phusion polymerase, 0.5 ⁇ 1; cDNA, 2 ⁇ 1.
  • the amplified product was ligated to a commercial cloning vector (pEASY-Blunt Simple) and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing.
  • a commercial cloning vector pEASY-Blunt Simple
  • the BALB/C mouse system was used as a model to detect changes in cell function after overexpression of CD28 molecules.
  • the specific steps are as follows:
  • CD28 mRNA The synthesis of CD28 mRNA was carried out in accordance with the instructions of Promega's RiboMAXTM Large Scale RNA Production Systems-T7. After CD28 mRNA synthesis, the RNA was purified using the RNessy Mini Kit (Qiagen), and finally the RNA was dissolved in DEPC water, and the concentration was measured by a spectrophotometer and stored at -80 °C.
  • B7 molecule (recombinant B7-1/Fc chimeric protein (R&D Systems, Minneapolis, MN)) was also diluted to 0.45 (pH 7.2-7.4) to 0.4 ⁇
  • AMAXA AMAXA
  • the nuclear rotor was placed in a nuclear transfer instrument (AMAXA), transfected with the X001 program, and then rapidly added to 0.5 ml of 37 °C pre-warmed complete 1640 medium, and the cells were added to the anti-CD3 antibody and B7 molecule coated.
  • treated cell culture plates (about 0.7xl0 6 cells / well) were stimulated. The plates were incubated at 37 ° C in a 5% CO 2 cell culture incubator.
  • Detection of activation of murine spleen T cells after stimulation by antigen-presenting system Transfected T cells stimulated by a simulated antigen presentation system (CD3 antibody and B7 molecular coating system) 24 After an hour, FITC-labeled anti-mouse CD25 antibody, APC-labeled anti-murine CD44 antibody and PerCP/Cy5.5-labeled anti-mouse CD69 antibody (Biolegend) were stained, followed by flow cytometry for CD25, CD44 and CD69. The marker expression level is activated for detection.
  • cytokines IFN- ⁇ and IL-4
  • Transfected T cells subjected to simulated antigen presentation system ( After 48 hours of stimulation with CD3 antibody and B7 molecular coating system, the cell culture supernatant was collected and used.
  • the ELISA method quantifies the secreted cytokines.
  • the measurement of IFN- ⁇ was carried out in accordance with eBioscience's Mouse IFN- ⁇ ELISA kit instructions.
  • the measurement of IL-4 was carried out in accordance with Southern Biotech's Rat Anti-Mouse Interleukin-4 (IL-4) ELISA Set instruction.
  • Beta-Actin gene was set as an internal reference gene.
  • the primers used in the reaction are shown in the table below.
  • T-bet 5' TCATCACTAAGCAAGGACGG 3' 5' GACCACATCCACAAACATCC 3'
  • the plasmid containing the porcine CD28 gene is transfected into porcine peripheral blood mononuclear cells (PBMC, mainly containing T lymphocytes), and the cells are activated and activated at the cellular level when stimulated by antigen (PRRSV). Factor secretion activity.
  • PBMC porcine peripheral blood mononuclear cells
  • PRRSV antigen
  • GTAGGCTGCAAAG 3' (shaded portion is the HA tag sequence), and the porcine CD28 fragment was amplified using a cloning vector containing the CD28 gene as a template.
  • the reaction system and procedure are the same as those in Example 1.
  • the cloned fragments contained EcoRI and BamHI restriction sites at both ends, and the HA tag sequence was introduced downstream to facilitate the detection of fusion protein expression by western-blot.
  • the amplified fragment was ligated to the backbone vector pIRES2-EGFP (purchased from BD Biosciences Clontech) co-digested with EcoRI and BamHI.
  • the constructed plasmid is shown in Figure 1.
  • ⁇ 10 ⁇ PMBC was dissolved in mouse lymphocyte transfection kit ( ⁇ , mouse T cell transfection kit), and 4 ⁇ pIRES-CD28HA was added, and a negative group was established.
  • Eestem-blot method was used to detect the expression of foreign genes. Due to the vector pIRES-CD28HA used for transfection, the CD28 gene carried by the vector has been fused to the HA tag, so that the commercially available antibody against the HA-anti-HA tag can be hybridized with the fusion protein and labeled with HRP (horseradish peroxidase). The anti-mouse secondary antibody was tested for hybridization.
  • HRP horseradish peroxidase
  • PBMC peripheral blood mononuclear cells
  • PRRSV antigen
  • the transcription level of the T cell-activated Marker CD25 molecule was analyzed, and the GAPDH gene was set as an internal reference gene.
  • the primers used are shown in Table 3 below.
  • PBMC peripheral blood mononuclear cells
  • PRRSV antigen
  • the co-stimulatory receptor CD28 provided by the present invention is specifically expressed in T cells, and can enhance the activation, proliferation and cytokine secretion activity of T cells when stimulated by antigen, thereby enhancing the host immune response and enhancing the vaccine immune effect.

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Abstract

提供了猪CD28受体分子，其为：1）由SEQ ID NO：2所示氨基酸序列组成的蛋白质，或2）在SEQ ID NO：2所示氨基酸序列中经取代、缺失或添加一个或几个氨基酸，且具有同等活性的由1）衍生的蛋白质。还提供了猪CD28受体的编码基因，其核苷酸序列如SEQ ID NO：1所示。当所提供的共刺激受体CD28特异性的高表达于T细胞时，可以增强T细胞受到抗原刺激时的激活、增殖和细胞因子分泌活性，进而增强宿主的获得性免疫应答，增强疫苗的免疫效果。

Description

猪 CD28受体， 编码其的基因及其应用 技术领域
本发明属于动物基因工程领域， 具体涉及猪 CD28受体， 编码其的基因及 其应用。 背景技术
中国是一个猪肉生产和消费大国，然而目前猪瘟、繁殖和呼吸综合症（蓝 耳病， PRRSV ) 等疫病严重流行是我国养猪行业面临的最大困难， 因此疫病 防控成为稳定和发展养猪业亟待解决的重大问题。 然而， 近年来猪病普遍呈 现多病原交叉混合感染， 猪的抗病能力和疫苗的防治效果明显降低。 因此要 做好疫病防控工作， 需要寻找疫苗研发的新策略， 或者寻求替代或辅助途径， 如培育出具有抗病性的新品种猪。 然而已有的研究成果多致力于利用特定疾 病抗性基因培养单一抗病特性的家畜新品种。 而这种策略显然与当前我国养 猪业疫病多发的现实状况差距太大。 因此， 培育出具有广谱抗病性的新品种 猪成为众多科研人员的理想。
适应性免疫应答反应在动物抵抗病原体侵袭过程中发挥着至关重要的作 用。 T细胞的活化更是该反应的重中之重。 近年来的研究证明， T细胞的有效 激活需要双信号刺激。 一是 MHC-抗原肽与 TCR结合产生的第一信号， 二是共 刺激受体， 主要是 CD28与其配体结合产生的第二信号。 在正常的机体微环境 下， 抗原递呈细胞递呈出来的抗原肽往往数量较少， 其与 TCR的结合不足以 活化 T细胞， 容易造成 T细胞反应无能。 而 T细胞共刺激受体 CD28等提供的共 刺激信号通路可以弥补较弱的 TCR信号， 从而激活 T细胞。 再者， 当 TCR与抗 原肽的亲和不足时，往往难以活化 T细胞， 而足够的共刺激信号也能起到增强 TCR信号通路的作用， 从而激活 T细胞。 总之， 足够的共刺激信号， 既可以克 服 TCR占有率较少的问题， 还可以弥补 TCR亲和力的不足。 因此共刺激受体 介导的信号能增强适应性免疫系统功能， 从而具备成为广谱抗病基因候选的 潜能。
CD28受体作为共刺激受体中的典型代表， 其基因序列信息和蛋白功能在 人和小鼠的相关研究中， 已有详尽的描述。 CD28受体是公认的 T细胞起始增 殖及存活的主要共刺激分子。 该受体与其配体 B7.1 (亦名 CD80 )或 B7.2 (亦 名 CD86 )连接后， 对 T细胞的激活、 增殖和存活都有明显的促进作用， 而且 可以影响 T细胞的分化方向， 并上调 IFN-γ等细胞因子的表达。 已有研究发现 老年人 T细胞表面的 CD28分子表达水平明显下调， 某些免疫系统激活滞后的 病例也表现为 CD28分子表达水平较低。 因此， CD28分子的表达水平直接影 响免疫系统的有效激活， 进而影响生物个体的抗病能力。
CD28受体对 T细胞免疫反应的建立、 增强及其维持都具有重要作用， 这 预示着他们作为靶基因在转基因育种研究及应用中具有良好的前景。 但是目 前绝大多数共刺激分子相关研究都是基于基因敲除或者是利用抗 CD28的单 抗来除去或者增强共刺激信号的方式进行的。这些策略明显不适于育种研究。 再者， 猪的 CD28受体目前只有预测的基因序列信息被公布， 其确切的编码序 列及其蛋白功能在此之前均未有报道。 发明内容
本发明的目的在于提供一种猪 CD28受体， 编码其的基因及其应用。
为实现上述目的，本发明首先提供一种猪 CD28受体，其为：由 SEQ ID NO. 2所示的氨基酸序列组成的蛋白质。
应当理解， 本领域技术人员可根据本发明公开的氨基酸序列， 在不影响 其活性的前提下， 取代、 缺失和 /或增加一个或几个氨基酸， 得到所述蛋白的 突变序列。 因此， 本发明猪 CD28受体还包括由 SEQ ID N0. 2所示的氨基酸序 列中经取代、 缺失或添加一个或几个氨基酸且具有同等活性的由 SEQ ID NO. 2所示的蛋白质衍生的蛋白质。 例如在非活性区段， 将第 181位的丝氨酸替换 为苏氨酸，或是将第 188位的谷氨酰胺缺失，或是在 198位后面增加 3个脯氨酸。
优选的，衍生蛋白的氨基酸序列与 SEQ ID No.2所示的氨基酸序列的同源 性可为 70%以上， 优选 80%以上， 更优选 90%以上。
本发明还提供编码上述蛋白的基因。 优选的， 本发明提供的编码猪 CD28 受体的基因的核苷酸序列如 SEQ ID No.l所示。
应理解， 考虑到密码子的简并性以及不同物种密码子的偏爱性， 本领域 技术人员可以根据需要使用适合特定物种表达的密码子。
本发明还提供的含有猪 CD28受体基因的载体、细胞系及宿主菌均属于本 发明的保护范围。
本发明还提供所述的猪 CD28受体在提高猪广谱抗病性中的应用。 如通过 制备抗猪 CD28单抗来研制生物制剂， 以口服或注射方式给药后， 该单抗与 CD28受体的结合可增强 T细胞激活所需的第二信号， 进而增强 T细胞免疫反 应， 提高猪的抗病能力。
本发明还提供上述的猪 CD28受体基因在培育广谱抗病性猪中的应用。 具 体方法为：
1 )将猪 CD28受体基因克隆到真核表达载体上， 或通过体外转录的方法获 得猪 CD28受体基因 mRNA;
2 )利用电穿孔法将制备的重组载体或 mRNA导入猪胚胎细胞；
3 )获得 CD28表达水平上调， 从而广谱性抗病性提高的转基因猪。
本发明基于共刺激受体 CD28的转基因策略是动物育种研究的一项新型 策略。将共刺激受体 CD28特异性地高表达于 T细胞， 可以增强 T细胞在受到抗 原刺激时的激活、增殖和细胞因子分泌活性， 进而增强宿主后天性免疫应答、 增强疫苗免疫效果。 所以， 本领域技术人员可以预见转基因动物个体培育成 功后， 在病原感染时的免疫功能及其抗病能力也将明显提高。 基于高表达 CD28的策略， 与培养单一抗病特性的家畜新品种相比， 可以更有效地地增强 宿主在受到多病原交叉混合感染时的抗病能力， 并提高疫苗效果。 附图说明
图 1是载体 pIRES-CD28HA构成模式图， 其中 CMV: 巨细胞病毒（CMV ) 启动子， 可调控目的基因在真核细胞内表达的启动子； CD28:猪 CD28基因编 码区； HA: 编码一个 HA (流感病毒血细胞凝集素抗原决定簇)短肽的一段基 因序列， 可作为标签来检测所融合蛋白的表达情况； IRES: 真核 mRNA 5'端 具有一段较短的 RNA序列， 能独立地起始翻译； EGFP: 增强型绿荧光蛋白基 因编码区； SV40: 是是猴空泡病毒 40 mRNA末端的一段多聚腺苷酸残基， 具 有终止转录、 增强 RNA的稳定性的作用。 图 2是用鼠 CD28 mRNA转染鼠 T细胞后， 用流式细胞术检测其 CD28高表 达的情况。其中 Α为转染 2(^g CD28 mRNA后，高表达 CD28的 T细胞数量增多； B为高表达 CD28的 T细胞荧光强度增强，即平均每个 T细胞表面 CD28分子数量 增多。
图 3是用鼠 CD28 mRNA转染鼠 T细胞后，在受到抗原递呈系统刺激时， 细 胞表面激活标志分子（ Marker )表达水平上调的情况。其中 A为转染 20 g CD28 mRNA后， 激活 marker ( CD25、 CD44和 CD69 ) 高表达的 T细胞数量增多； B 为高表达激活 Marker ( CD25、 CD44和 CD69 ) 的 T细胞荧光强度增强， 即平 均每个 T细胞表面激活 Marker数量增多。
图 4是用鼠 CD28 mRNA转染鼠 T细胞后，在受到抗原递呈系统刺激时， 细 胞分泌 IFN-γ的量增多，但 IL-4的分泌量较未转染 CD28 mRNA的细胞没有明显 变化。
图 5是用鼠 CD28 mRNA转染鼠 T细胞后，在受到抗原递呈系统刺激时， 细 胞分化方向受到明显影响。 其中转录因子 T-bet， GATA3 , RORyt的上调表明 更多 T细胞向 Thl , Th2和 TM7这些具有正向免疫调控作用的亚型分化， Foxp3 因子的下调表明 T细胞向 Treg方向 （负向调控） 的分化受到抑制。
图 6是猪外周血单个核细胞 （PBMC ) 转染 pIRES-CD28HA质粒后， 用 Western blot方法检测其外源蛋白表达情况。
图 7是猪外周血单个核细胞 （PBMC ) 转染 pIRES-CD28HA质粒后在受到 抗原（PRRSV )刺激时， 其 T细胞激活 marker CD25分子的转录水平明显提高。
图 8是猪外周血单个核细胞 （PBMC ) 转染 pIRES-CD28HA质粒后在受到 抗原 （PRRSV )刺激时， 其 PBMC细胞内 IFN-γ转录水平明显提高。 具体实施方式
以下实施例用于说明本发明， 但不用来限制本发明的范围。 实施例 1 CD28基因的克隆
本发明以人和猪基因序列比对结果具有较高相似性为理论基础， 根据人 的 CD28序列设计引物， 以五指山猪的 cDNA为模板调取猪的 CD28疑似序 列。 具体操作步骤为： ( 1 )通过与人类的 CD28基因序列进行比对， 在猪的基因组中找到相似 性较高的基因片段， 并据此设计引物 （见表 1 : P1和 P2 )。
( 2 )猪外周血单个核细胞 （PBMC ) cDNA的提取：
从猪的前腔静脉无菌釆取 20ml肝素钠抗凝血， 经 PBS等体积稀释、 混 匀后， 将其缓慢加于等体积的猪淋巴细胞分离液 （天津灏翔， 中国）上， 水 平转子离心 1800rpm , 20min。 之后吸取血浆下的淋巴细胞层， 获得 PBMC。 利用 Invitrogen公司的 Trizol试剂（ TRIzol® LS Reagent )提取 PBMC 的总 RNA, 再用 ABI公司的反转录试剂盒（First Strand Synthesis Kit for RT-PCR )合成猪的 cDNA。
( 3 ) 以猪的 cDNA为模板， 利用 NEB公司的 phusion 高保真聚合酶试 剂盒扩增其 CD28的疑似序列。
扩增反应体系： 双蒸水， 34.5μ1; 5 xHF buffer, ΙΟμΙ; lOmM dNTP mix, Ιμΐ; 25μΜ Ρ1, Ιμΐ; 25μΜ Ρ2, Ιμΐ; phusion聚合酶， 0.5μ1; cDNA, 2μ1。
反应程序： 预变性：98°C,lmin;
循环 (35个)： 98°C, 10s; 55°C, 20s; 72°C, lmin;
补充延伸： 72°C， 10min。
扩增所得产物连接到商品化克隆载体（pEASY-Blunt Simple ) 上， 送至 上海美吉生物医药科技有限公司测序。
( 4 )根据步骤（3 )所测得的序列信息设计 5'RACE和 3'RACE所需引 物 （表 1 : 5'GSPl和 5，GSP2; 3'GSPl和 3，GSP2 ), 利用 TAKARA公司的 试剂盒（ 5，-Full RACE Kit 和 3，-Full RACE Core Set Ver.2.0 )， 获取其 5，和 3， 末端的转录非翻译区 （UTR )序列信息， cDNA全长序列如 SEQ ID No.l所 示， 该蛋白的全长氨基酸序列如 SEQ ID No.2所示。
表 1猪 CD28基因克隆所用的引物
用途 引物名称 引物序列
初步扩增猪 CD28序歹1 J PI 5' ATGCTCAGGCTGCTCTTGGCTC 3'
P2 5' TCAGGAGCGATAGGCTGCGAAG 3'
5'RACE 5'GSPl 5'ACATTCACAACACAGACCTCCACAG 3'
5'GSP2 5' GGTTGTAGGTGTACTTGCAGCTAAG 3'
3'RACE 3'GSPl 5' TGGTGGTGGTAAATGGAGTCGT 3'
3 'GSP2 5' GAGTGACTACATGAACATGACC 3' 实施例 2 小鼠模型中 CD28基因上调表达对 T细胞免疫反应的影响
以 BALB/C小鼠系统为模型， 检测其 CD28分子过表达后的细胞功能变 化情况。 具体操作步骤为：
( 1 )质粒 pGEM4Z/mCD28/A64的构建： 以 pGEM4Z (购自 Promega ) 为出发载体， 按照 David Boczkowski, Smita K. Nair, Jong-Hee Nam, et al.， Induction of Tumor Immunity and Cytotoxic T Lymphocyte Responses Using Dendritic Cells Transfected with Messenger RNA Amplified from Tumor Cells, 2000公开的方法构建质粒 pGEM4Z/mCD28/A64。
( 2 ) CD28 mRNA的合成与纯化： CD28 mRNA的合成遵照 Promega公 司的 RiboMAX™ Large Scale RNA Production Systems-T7的说明书进行。 CD28 mRNA合成后， 用试剂盒 RNessy Mini Kit ( Qiagen ) 纯化 RNA, 最 后将 RNA溶于 DEPC水中， 分光光度计测定浓度后分装保存于 -80°C。
( 3 )模拟抗原递呈系统的建立（即在 96 孔细胞培养板上包被抗 CD3 抗体和 B7 分子） ： 将抗鼠 CD3 抗体（Anti-murine CD3e， BD ) 用 PBS
( pH7.2-7.4 )稀释为 2 g/ml， B7分子 (recombinant B7-1/Fc chimeric protein (R&D Systems, Minneapolis, MN))也用 PBS ( pH7.2-7.4 )稀释为 0.4 μ^ιηΐ, 向每个细胞孔中加入稀释后的抗 CD3抗体和 Β7分子各 50μ1， 4。C过夜孵育 平板。 用 PBS洗涤两次后， 可向孔中加入 T细胞。
( 4 )鼠脾脏 T细胞提取： 遵照 Miltenyi Biotec公司的 Pan T Cell Isolation Kit说明书进行。
( 5 ) 鼠脾脏 T细胞的转染： 将分离的到的 2xl06 T细胞溶于 ΙΟΟμΙ鼠 Τ 细胞转染液（ Nucleofector® Solution, AMAXA )， 加入 20μ§ CD28 mRNA, 同时设立阴性对照， 即不加 RNA， 轻轻混匀后转移到 2mm 核转杯
( AMAXA ) 中。 将核转杯放入核转仪（AMAXA )中， 用 X001程序进行转 染， 然后迅速加入 0.5ml 37°C预热的完全 1640培养基， 并将细胞加入抗 CD3 抗体和 B7分子包被处理过的细胞培养板上（约 0.7xl06细胞 /孔）进行刺激。 将培养板置于 37°C， 5%C02的细胞培养箱中培养。
( 6 ) 鼠脾脏 T细胞转染后 CD28表达水平的检测： 转染后的 T细胞， 受模拟抗原递呈系统 （CD3抗体和 B7分子包被体系）刺激 24小时后， 用 APC标记的抗鼠 CD28 抗体 (Biolegend)进行染色， 再用流式细胞术对 CD28 的表达水平进行检测。
( 7 ) 鼠脾脏 T细胞转染后在受到抗原递呈系统刺激时， 其激活情况的 检测： 转染后的 T细胞， 受模拟抗原递呈系统（CD3抗体和 B7分子包被体 系）刺激 24小时后， 用 FITC标记的抗鼠 CD25抗体， APC标记的抗鼠 CD44 抗体和 PerCP/Cy5.5标记的抗鼠 CD69抗体 (Biolegend)进行染色， 再用流式细 胞术对 CD25, CD44和 CD69等激活 marker的表达水平进行检测。
( 8 ) 鼠脾脏 T 细胞转染后在受到抗原递呈系统刺激时， 其细胞因子 ( IFN-γ和 IL-4 )分泌情况的检测： 转染后的 T细胞， 受模拟抗原递呈系统 ( CD3抗体和 B7分子包被体系 )刺激 48小时后， 收集细胞培养上清， 用
ELISA 方法对所分泌的细胞因子进行定量分析。 IFN-γ 的测定是参照 eBioscience公司的 Mouse IFN-λ ELISA kit说明书进行。 IL-4的测定是参照 SouthernBiotech公司的 Rat Anti-Mouse Interleukin-4 (IL-4) ELISA Set说明书 进行。
( 9 ) 鼠脾脏 T细胞转染后在受到抗原递呈系统刺激时， 其分化方向的 检测： 转染后的 Τ细胞， 受模拟抗原递呈系统（CD3抗体和 Β7分子包被体 系）刺激 48小时后， 提取细胞 RNA, 并进行反转录。 用相对荧光定量 PCR
( SYBGreen染料法）对 T-bet， GATA3 , RORyt和 Foxp3等转录因子的转录 水平进行分析， 并设置 Beta-Actin基因为内参基因。 反应所用的引物见下 表。
表 2 用相对荧光定量 PCR (SYBGreen染料法） 对鼠各转录因子的转录水平进行分析时 所用的引物
基因 上游引物 下游引物
T-bet 5' TCATCACTAAGCAAGGACGG 3' 5' GACCACATCCACAAACATCC 3'
Gata3 5' GTCCTCATCTCTTCACCTTCC 3' 5， CACTCTTTCTCATCTTGCCTG 3'
RORyt 5， CAAGTTCTCAGTCATGAGAACAC 3' 5， GAGTAGGCCACATTACACTG 3'
Foxp3 5， TTCCTTCCCAGAGTTCTTCC 3' 5， GGTAGATTTCATTGAGTGTCCT 3'
Beta-Actin 5' CATCACTATTGGCAACGAGC 3' 5， GACAGCACTGTGTTGGCATA 3， 结果表明， 用 mRNA转染 T细胞后， 高 APC荧光强度的细胞数量明显 增多， 即 CD28表达水平明显上调 （图 2 )。 高表达 CD28的 T细胞在受到抗 原递呈系统刺激时， 其细胞表面的激活标志分子 CD25， CD44和 CD69的表 达水平均明显上调（图 3 )。 ELISA结果显示， 转染 CD28mRNA后的 T细胞 在受到刺激后， 其细胞因子 IFN-γ的分泌活性也明显增强 （图 4 )。 而且， 据 转录水平的检测结果， 其 T-bet ( Thl型）， GATA3 ( Th2型）， RORyt ( Thl7 型）转录因子的表达水平明显上调， 表明 T细胞向 Thl， Th2和 Thl7这些具 有正向免疫调控作用的亚型分化趋势增强 （图 5 )。 实施例 3 猪 CD28基因上调表达对猪 T细胞免疫反应的影响
利用本发明提供的策略， 将含有猪 CD28基因的质粒转染猪外周血单个 核细胞 （PBMC, 主要包含 T 淋巴细胞） ， 在细胞水平上检测其受到抗原 ( PRRSV )刺激时细胞的激活和细胞因子分泌活性。
( 1 ) 真核表达载体 pIRES-CD28HA 的构建。 设计上游引物： 5' (EcoRI)GAATTCATGATCCTCGGGTTACTCCTGG 3'和 下 游 引 物 ： 5，
GTAGGCTGCAAAG 3' (阴影部分为 HA标签序列）， 以含 CD28基因的克隆 载体为模板， 扩增猪 CD28片段。 反应体系和程序同实施例 1 中的步骤 3。 所克隆的片段两端分别含有 EcoRI和 BamHI酶切位点，且下游引入了 HA标 签序列， 便于用 western-blot检测融合蛋白表达情况。 最后， 将扩增所得的片 段连接到经 EcoRI和 BamHI共酶切后的骨架载体 pIRES2-EGFP (购自 BD Biosciences Clontech ) 上。 构建后的质粒见图 1。
( 2 ) pIRES-CD28HA质粒的大量提取：
参照 OMEGA公司生产的 E.Z.N.A.TM Endo-Free Plsamid Maxi Kit说明 书进行。 质粒提取完成后， 用分光光度计测定质粒的浓度。
( 3 )猪外周血单个核细胞（PBMC ) 的提取：
从猪的前腔静脉无菌采取 20ml肝素钠抗凝血， 经 PBS等体积稀释、 混 匀后， 将其缓慢加于等体积的猪淋巴细胞分离液（天津灏翔， 中国） 上， 水 平转子离心 1800rpm , 20min。 之后吸取血浆下的淋巴细胞层， 获得 PBM (：。 ( 4 )猪外周血 PBMC的转染：
细胞计数后， 将 5χ 10ό PMBC溶于 ΙΟΟμΙ恢复至室温的鼠淋巴细胞转染 缓冲液中 （ ΑΜΑΧΑ, mouse T cell transfection kit ) , 力口入 4μ§ pIRES-CD28HA, 同时设立阴性组， 即不加质粒， 轻轻混匀后转移到 2mm 核转杯 （AMAXA ) 中。 将核转杯放入核转仪 （AMAXA ) 中， 用核转仪 ( AMAXA LONZA ) 中的固有程序 Z001程序进行转染， 然后迅速将细胞转 移至 2ml 37°C预热的完全 1640培养基中， 并置于 37°C， 5%C02的细胞培养 箱中培养。
( 5 )用 Eestem-blot方法检测外源基因的表达情况。 因转染所用的载体 pIRES-CD28HA, 其携带的 CD28基因已与 HA标签融合， 故可用鼠源抗 HA 标签的商品化抗体与融合蛋白杂交， 再用 HRP (辣根过氧化物酶）标记的抗 鼠二抗对杂交情况进行检测。
( 6 )猪外周血单个核细胞（PBMC )转染后在受到抗原（PRRSV )刺激 时， 其激活情况的检测： 用载体pIRES-CD28HA转染猪PBMC 8小时后， 用 0.1MOI PRRSV 感染这些细胞。 感染 24 小时后， 利用相对荧光定量 PCR
( SYBGreen染料法）对其 T细胞激活 Marker CD25分子的转录水平进行分 析， 并设置 GAPDH基因为内参基因。 所用引物见下表 3。
表 3 用相对荧光定量 PCR ( SYBGreen染料法）对猪的激活 Marker (CD25分子）和 IFN-γ
的转录水平进行分析时所用的引物
基因 上游引物 下游引物
CD25 5， CTAATCTTCCAGGTCACTGC 3' 5， CCTCCATGAAGTGGTAAACTC 3'
IFN-γ 5， GCAAGTACCTCAGATGTACCT 3' 5' TTGTCACTCTCCTCTTTCCA 3'
GAPDH 5， CTCAACGGGAAGCTCACTGG 3' 5' TGATGTCATCATATTTTGCAGGTT 3'
( 7 )猪外周血单个核细胞（PBMC )转染后在受到抗原（PRRSV )刺激 时， 其 IFN-γ转录水平的检测： 用载体 11 £8 0281^转染猪？8]\^ 8小时 后， 用 0.1MOI PRRSV感染这些细胞。 感染 24小时后， 利用相对荧光定量 PCR ( SYBGreen染料法）对 PBMC细胞内 IFN-γ转录水平进行分析， 并设 置 GAPDH基因为内参基因。 所用引物见表 3。
结果如附图所示， 载体 pIRES-CD28HA转染猪 PBMC细胞后， 用抗 HA单 抗检测到融合蛋白在细胞内可成功表达（图 6 )。 高表达 CD28的 PBMC细胞在 受到感染 PRRSV的递呈细胞刺激后，其激活标志分子 CD25的转录水平明显上 调 （图 7 )， 激活的 PBMC其细胞因子 IFN-γ的转录水平（图 8 )也明显提高。 工业实用性
本发明提供的共刺激受体 CD28特异性地高表达于 T细胞， 可以增强 T细 胞在受到抗原刺激时的激活、 增殖和细胞因子分泌活性， 进而增强宿主后天 性免疫应答、 增强疫苗免疫效果。

Claims

权 利 要 求 书
1、 猪 CD28受体分子， 其为：
1 ) 由 SEQ ID NO. 2所示的氨基酸序列组成的蛋白质， 或
2 )在 SEQ ID NO. 2所示的氨基酸序列中经取代、 缺失或添加一个或几 个氨基酸且具有同等活性的由 1 )衍生的蛋白质。
2、 编码权利要求 1所述的猪 CD28受体的基因。
3、根据权利要求 2所述的基因，其特征在于，核苷酸序列如 SEQ ID NO.
1所示。
4、 含有杈利要求 2或 3所述基因的载体。
5、 含有权利要求 4所述载体的宿主细胞。
6、 权利要求 1所述的猪 CD28受体在提高猪广谱抗病性中的应用。
7、 根据权利要求 6所述的应用， 其特征在于， 以权利要求 1 所述的猪 CD28受体分子为免疫原， 制备 CD28的单克隆抗体， 再制备成制剂后进行给 药， 提高猪的广谱抗病能力。
8、 权利要求 2或 3所述的基因在培育广谱抗病性猪中的应用。
9、 根据权利要求 8所述的应用， 其特征在于， 包括如下步骤：
1 )将猪 CD28受体基因克隆到真核表达载体上， 或通过体外转录的方法 获得猪 CD28受体基因 mRNA;
2 )利用电穿孔法将制备的重组载体或 mRNA导入猪胚胎细胞；
3 )获得 CD28表达水平上调， 从而广谱性抗病性提高的转基因猪。
I I
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