WO2021217959A1 - 一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用 - Google Patents

一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用 Download PDF

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WO2021217959A1
WO2021217959A1 PCT/CN2020/109703 CN2020109703W WO2021217959A1 WO 2021217959 A1 WO2021217959 A1 WO 2021217959A1 CN 2020109703 W CN2020109703 W CN 2020109703W WO 2021217959 A1 WO2021217959 A1 WO 2021217959A1
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swine fever
african swine
recombinant
protein
fever virus
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PCT/CN2020/109703
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French (fr)
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董伟
谢小红
文利新
吴朝亮
李鑫
朱圆缘
张永勇
黄佳豪
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长沙绿叶生物科技有限公司
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Priority to RU2021116273A priority Critical patent/RU2771177C1/ru
Priority to US17/299,375 priority patent/US20220315947A1/en
Priority to EP20891466.3A priority patent/EP3922724A4/en
Publication of WO2021217959A1 publication Critical patent/WO2021217959A1/zh

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Definitions

  • the present invention belongs to the technical field of gene recombination, and specifically relates to a recombinant vector, recombinant bacteria and application thereof containing the immunogenic protein of African swine fever virus.
  • African swine fever is an acute, febrile, and highly contact animal infectious disease. Its incidence and lethality can be as high as 100%. It is the number one killer in the pig industry. There is currently no commercial vaccine available.
  • African swine fever virus African swine fevervirus, ASFV
  • ASFV African swine fevervirus
  • the virus particle has a diameter of 175 to 215 nanometers and is icosahedral.
  • the nucleocapsid is covered with a capsule;
  • the genome is a double-stranded linear DNA with a size between 170 and 190 kb.
  • African swine fever virus can cause high-contact transmission between domestic pigs and various wild boars. It enters the pig body mainly through the mouth and upper respiratory system. Infection occurs in the nasopharynx or tonsils. The virus quickly spreads to the mandibular lymph nodes. Blood invades the whole body.
  • biosafety prevention and control Since there is no commercial vaccine for ASFV on the market, the safest, most economical, and most effective prevention and control method is biosafety prevention and control.
  • the principle of prevention and control is to block the virus from contacting the body, but the existing methods cannot guarantee it. The virus and the body are no longer in contact.
  • the purpose of the present invention is to provide a recombinant vector containing the immunogenic protein of African swine fever virus, recombinant bacteria and their applications, and to construct a Lactobacillus expression system expressing the p72 and p54 proteins of African swine fever virus for research purposes.
  • a Lactobacillus expression system expressing the p72 and p54 proteins of African swine fever virus for research purposes.
  • the present invention provides a recombinant vector containing the immunogenic protein of African swine fever virus.
  • the recombinant vector uses the Lactobacillus expression vector pVE5523 as the basic vector to clone the nucleotide sequence encoding the immunogenic protein of African swine fever virus. To the EcoRV and SalI restriction sites of the basic vector.
  • the immunogenic protein of the African swine fever virus includes the p72 protein and the p54 protein of the Chinese Jilin strain of African swine fever virus; the nucleotide sequence encoding the p72 protein of the Chinese Jilin strain of African swine fever virus is as SEQ ID NO.1 As shown; the nucleotide sequence encoding the p54 protein of the Chinese Jilin strain of African swine fever virus is shown in SEQ ID NO.2.
  • the present invention provides a recombinant Lactobacillus expressing an immunogenic protein of African swine fever virus, and the recombinant Lactobacillus comprises the recombinant vector.
  • the present invention also provides a method for constructing the recombinant Lactobacillus, which includes the following steps: (1) The nucleotide sequence encoding the p72 protein of African swine fever virus and the nucleotide sequence encoding the p54 protein of African swine fever virus are respectively cloned into In the pVE5523 Lactobacillus expression vector, construct recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54;
  • the recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54 were transformed into Lactobacillus competent, respectively, to obtain recombinant Lactobacillus expressing African swine fever virus p72 protein and recombinant Lactobacillus expressing African swine fever virus p54 protein .
  • the present invention also provides an oral live bacterial preparation for preventing African swine fever infection.
  • the effective ingredients of the oral live bacterial preparation include: recombinant lactobacillus expressing the p72 protein of African swine fever virus constructed by the construction method and expression Recombinant Lactobacillus of African swine fever virus p54 protein.
  • the ratio of viable bacteria of the recombinant Lactobacillus expressing the p72 protein of African swine fever virus and the recombinant Lactobacillus expressing the p54 protein of African swine fever virus is (0.8 ⁇ 1.2) ⁇ 10 8 cfu: (0.8 ⁇ 1.2) ⁇ 10 8 cfu.
  • the present invention provides a recombinant vector containing the immunogenic protein of African swine fever virus.
  • the recombinant vector can be used to construct a recombinant lactobacillus expressing the immunogenic protein of African swine fever virus. After the p72 and p54 recombinant lactobacilli are mixed, an oral viable preparation that can be used to prevent African swine fever virus infection is prepared.
  • the antigen protein (mixture of protein p72 and p54) secreted by the lactobacillus in the preparation adheres to the cell surface mucosa of the body, and forms an antigen protein biofilm on the mucosal surface, and the antigen protein and target cells
  • the binding sites on the virus bind to the site of the virus receptor protein on the mucosal surface and play an ecological role.
  • the virus binding site on the target cell has been affected by the antigen protein organism.
  • the membrane is completely sealed, and the virus cannot bind to the virus binding site on the target cell, thereby effectively blocking the binding of the virus to the receptor on the cell surface, and has the effect of preventing African swine fever.
  • the oral preparations created by the invention are safer, more effective and faster.
  • the safety of the oral agent of the present invention is shown in the fact that the active ingredients only secrete viral functional proteins without the presence of viral genes and will not cause virus mutation; the effectiveness is shown in the fact that the active ingredients of the oral agent only secrete protective antigens and act on The mucosal part of the body surface covers the mucosal surface where the target cells of the African swine fever virus are located.
  • the site where it binds to the mucosal surface cells is blocked and sealed, thereby blocking the path of virus infection; rapidity is expressed in Lactobacillus expression
  • the secreted protein directly preempts the receptor that the virus binds to the target cell to block the virus infection, without the need for an immune response process.
  • Figure 1 is a plasmid structure diagram of the recombinant vector pVE5523-ASFV-p72 of the present invention
  • Figure 2 is a plasmid structure diagram of the recombinant vector pVE5523-ASFV-p54 of the present invention
  • Figure 3 is an amplification curve for verifying the expression of p72 in the present invention.
  • Figure 4 is an amplification curve for verifying the expression of p54 in the present invention.
  • the present invention provides a recombinant vector containing the immunogenic protein of African swine fever virus.
  • the recombinant vector uses the Lactobacillus expression vector pVE5523 as the basic vector to clone the nucleotide sequence encoding the immunogenic protein of African swine fever virus. To the EcoRV and SalI restriction sites of the basic vector.
  • the immunogenic protein of the African swine fever virus of the present invention includes the p72 protein and the p54 protein of the Chinese Jilin strain of African swine fever virus; the nucleotide sequence encoding the p72 protein of the Chinese Jilin strain of African swine fever virus is shown in SEQ ID NO.1 Show; the nucleotide sequence encoding the p54 protein of the Chinese Jilin strain of African swine fever virus is shown in SEQ ID NO.2.
  • the present invention clones the nucleotide sequence shown in SEQ ID NO.1 between the EcoRV and SalI restriction sites of the basic vector to form the recombinant vector pVE5523-ASFV-p72.
  • the structure of the recombinant plasmid is shown in Figure 1 ;
  • the nucleotide sequence shown in SEQ ID NO. 2 is cloned into the EcoRV and SalI restriction sites of the basic vector to form a recombinant vector pVE5523-ASFV-p54.
  • the structure of the recombinant plasmid is shown in Figure 2.
  • the present invention does not specifically limit the construction method of the recombinant vector, and conventional recombinant vector construction methods can be used.
  • the present invention provides a recombinant Lactobacillus expressing an immunogenic protein of African swine fever virus, and the recombinant Lactobacillus comprises the recombinant vector.
  • the present invention also provides a method for constructing the recombinant Lactobacillus, which includes the following steps: (1) The nucleotide sequence encoding the p72 protein of African swine fever virus and the nucleotide sequence encoding the p54 protein of African swine fever virus are respectively cloned into In the pVE5523 Lactobacillus expression vector, construct recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54;
  • the recombinant plasmids pVE5523-ASFV-p72 and pVE5523-ASFV-p54 were transformed into Lactobacillus competent, respectively, to obtain recombinant Lactobacillus expressing African swine fever virus p72 protein and recombinant Lactobacillus expressing African swine fever virus p54 protein .
  • the present invention preferably optimizes the p72 and p54 gene sequences of the Chinese Jilin strain of African swine fever virus obtained in GenBank to form the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 before proceeding.
  • GenBank Chinese Jilin strain of African swine fever virus obtained in GenBank
  • the present invention preferably uses electrotransformation to transform the recombinant plasmid into ATCC393 Lactobacillus casei competent cells, so as to obtain the recombinant Lactobacillus.
  • the present invention preferably further includes amplifying the recombinant Lactobacillus in MRS liquid medium, extracting the recombinant plasmid, and performing fluorescence quantitative PCR detection.
  • the primers and amplification sequences used in the fluorescence quantitative PCR detection of the present invention are as follows:
  • p72 upstream primer (SEQ ID NO.3): AGTTCGGATGTCACAACGCTTG;
  • Downstream primer of p72 (SEQ ID NO.4): TTTGCTTTGGTGCGGCTTGT;
  • p54 upstream primer (SEQ ID NO.6): AGCCACTCCACAACCAGGTAC;
  • Downstream primer of p54 (SEQ ID NO.7): GCCCTCCAGTTGCCATGATTAG;
  • the recombinant lactobacillus prepared by the construction method according to the present invention secretes the recombinant African swine fever virus immunogenic proteins p72 and p54 according to the difference of the recombinant plasmids.
  • the present invention also provides an oral live bacterial preparation for preventing African swine fever infection.
  • the active ingredients of the oral live bacterial preparation include recombinant lactobacillus expressing the p72 protein of African swine fever virus constructed by the construction method and expressing African swine fever virus.
  • the oral live bacteria preparation of the present invention can secrete the recombinant African swine fever virus immunogenic protein p72 and p54 mixed bacteria, the recombinant lactobacillus expressing the African swine fever virus p72 protein and the recombinant African swine fever virus p54 protein expressing
  • the viable bacteria ratio of the recombinant Lactobacillus is preferably (0.8 to 1.2) ⁇ 10 8 cfu: (0.8 to 1.2) ⁇ 10 8 cfu, more preferably 1 ⁇ 10 8 cfu: 1 ⁇ 10 8 cfu.
  • the p54 protein of African swine fever virus exists in the inner envelope of the virus particle. It is one of the main structural proteins and strong immunogenic proteins of ASFV, and is involved in the adsorption and entry of the virus to target cells.
  • GenBank GenBank: MK189456.1
  • P54 GenBank: MK214679.1
  • the sequence was optimized, and the modified sequence was obtained by Nanjing GenScript Biotechnology Co., Ltd.
  • the company's entire gene synthesis the base sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.
  • Restriction endonucleases SalI and EcoRV were purchased from NEB, and Taq enzymes, dNTPs, DNA Marker DL2000, DL15000, Agarose Gel DNA Purification Kit, Mini BEST Plasmid Purification Kit were purchased from Dalian Bao Biological Company.
  • the cloning vector pVE5523 was provided by Nanjing GenScript Biotechnology Co., Ltd.
  • the small fragments of the cloning vector pVE5523 digested with SalI/EcoRV double enzymes were respectively ligated with the p72 and p54 gene fragments digested with the same double enzymes, and the plasmids were extracted after electrotransformation and sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing verification.
  • Recombinant plasmid sequencing results The recombinant plasmid was sequenced with the inserted p72 and p54 gene fragments after gene sequencing. The sequencing results were consistent with expectations, indicating that the synthesized p72 and p54 gene fragments were successfully inserted into the Lactobacillus vector pVE5523, and the recombinant plasmid was constructed Success, the positive plasmids were named pVE5523-ASFV-p72 and pVE5523-ASFV-p54.
  • Erythromycin was purchased from Baierdi Biotechnology Co., Ltd.
  • Electrotransformation of the target gene in Lactobacillus ATCC393 and screening of resistant strains The electrotransformed Lactobacillus ATCC393 was spread on the MRS solid culture plate containing 5 ⁇ g/ml erythromycin and cultured in an incubator at 30°C for 72h. The colonies on the plate were spotted and inoculated into MRS liquid medium containing 5 ⁇ g/ml erythromycin, incubated at 30°C for 72h; plasmids in the bacteria were extracted and identified by fluorescence quantitative PCR, and the primers and amplified sequences were identified as follows.
  • p72 upstream primer (SEQ ID NO.3): AGTTCGGATGTCACAACGCTTG;
  • Downstream primer of p72 (SEQ ID NO.4): TTTGCTTTGGTGCGGCTTGT;
  • p54 upstream primer (SEQ ID NO.6): AGCCACTCCACAACCAGGTAC;
  • Downstream primer of p54 (SEQ ID NO.7): GCCCTCCAGTTGCCATGATTAG;
  • Fluorescence quantitative PCR was used to detect the amplified recombinant plasmids.
  • the amplification curves are shown in Figures 3 to 4 respectively.
  • the positive recombinant plasmids have typical amplification curves with CT values ranging from 19 to 22.
  • the ATCC393 competence control has no amplification. curve. It shows that the recombinant plasmids pVE5523-ASFV-p54 and pVE5523-ASFV-p72 have been successfully transformed into ATCC393 competent cells.
  • the recombinant Lactobacillus expression system was inoculated into Lactobacillus MRS liquid medium with 1% inoculum, and the fermentation broth was harvested at 35°C for 72 hours.
  • Dilution Use a 1mL pipette to suck the well-mixed Lactobacillus suspension (sample to be tested) 0.5ml to 10 -1 in a test tube, this is a 10-fold dilution. Place the 10 -1 test tube on the test tube shaker to make the bacteria liquid mix thoroughly. Take another 1ml pipette and insert it into the 10 -1 test tube to blow the bacteria suspension back and forth three times, further disperse and mix the bacteria, use this pipette to suck 0.5 mL of the 10 -1 bacteria liquid into the 10 -2 test tube , This is a 100-fold dilution, and the rest can be deduced by analogy.
  • Colony forming unit (cfu) per milliliter average number of colonies in three replicates of the same dilution ⁇ dilution factor ⁇ 5.
  • the lactobacilli capable of secreting recombinant p72 and p54 proteins prepared in Example 4 were diluted to 0.8-1.2 ⁇ 10 8 cfu/ml bacterial solution, and mixed in a volume ratio of 1:1.
  • the mixed bacterial liquid was administered to SPF New Zealand rabbits and SD rats in the form of oral liquid.
  • the negative control group A was given the same volume of normal saline and the control group B was given the same volume of MRS culture solution.
  • Each group of New Zealand rabbits and SD rats Four rats each were observed for 2 weeks. None of the 24 animals had abnormal body temperature, allergies and other abnormalities. This indicates that the lactobacillus capable of secreting recombinant p72 and p54 proteins prepared by the present invention is safe and has no side effects.
  • the results of the experiment are shown in Table 1.
  • the number of pigs raised in the first area of the control group increased from 112 to 66, and 46 died, with a mortality rate of 41.07%; in the second area, the number of pigs was reduced from 45 to 45 after use because it was already in the disease stage before the test.
  • the test results show that the recombinant Lactobacillus preparation has a good effect.

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Abstract

提供一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用，涉及基因重组技术领域；利用所述重组载体可构建表达非洲猪瘟病毒免疫原性蛋白的重组乳酸杆菌，将能分别分泌蛋白p72和p54的乳酸杆菌菌液混合后，制备可用于预防非洲猪瘟的口服活菌制剂。利用所述口服活菌制剂可安全、有效、快速的预防非洲猪瘟病毒对猪的感染，且不包含免疫过程。

Description

一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用
本申请要求于2020年04月26日提交中国专利局、申请号为202010339345.5、发明名称为“一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于基因重组技术领域，具体涉及一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用。
背景技术
非洲猪瘟(African swine fever，ASF)是一种急性、热性、高度接触性动物传染病，其发病率和致死率可高达100％，是养猪业头号杀手，目前没有商品疫苗可用。非洲猪瘟病毒(African swine fevervirus，ASFV)是非洲猪瘟病毒科非洲猪瘟病毒属的唯一成员，病毒粒子的直径为175～215纳米，呈20面体对称，核衣壳外面被有囊膜；基因组为双股线状DNA，大小在170～190kb之间。
非洲猪瘟病毒可引起家猪和各种野猪的高度接触性传播，主要经过口和上呼吸道系统进入猪体，在鼻咽部或是扁桃体发生感染，病毒迅速蔓延到下颌淋巴结，通过淋巴液和血液侵入全身。
由于目前还没有针对ASFV的商品化疫苗上市，目前最安全、最经济、最有效的防控手段是生物安全防控，防控原则是阻断病毒与机体接触，但是现有的方法都不能保证病毒和机体不再接触。
发明内容
有鉴于此，本发明的目的在于提供一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用，构建表达非洲猪瘟病毒p72和p54蛋白的乳酸杆菌表达系统，为研制用于阻断病毒感染的黏膜感染阻断口服剂提供理论基础。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种包含非洲猪瘟病毒免疫原性蛋白的重组载体，所述重组载体以乳酸杆菌表达载体pVE5523为基础载体，分别将编码非洲猪瘟病毒免疫原性蛋白的核苷酸序列克隆到所述基础载体的EcoRV和SalI酶切位点之间。
优选的，所述非洲猪瘟病毒免疫原性蛋白包括中国吉林株非洲猪瘟病毒p72蛋白和p54蛋白；编码所述中国吉林株非洲猪瘟病毒p72蛋白的核苷酸序列如SEQ ID NO.1所示；编码所述中国吉林株非洲猪瘟病毒p54蛋白的核苷酸序列如SEQ ID NO.2所示。
本发明提供了一种表达非洲猪瘟病毒免疫原性蛋白的重组乳酸杆菌，所述重组乳酸杆菌包含所述重组载体。
本发明还提供了所述重组乳酸杆菌的构建方法，包括以下步骤：(1)分别将编码非洲猪瘟病毒p72蛋白的核苷酸序列和编码非洲猪瘟病毒p54蛋白的核苷酸序列克隆到pVE5523乳酸杆菌表达载体中，构建重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54；
(2)分别将重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54转化至乳酸杆菌感受态中，得到表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌。
本发明还提供了一种预防非洲猪瘟感染的口服活菌制剂，所述口服活菌制剂的有效成分包括：利用所述构建方法构建得到的表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌。
优选的，所述表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌的活菌比为(0.8～1.2)×10 8cfu:(0.8～1.2)×10 8cfu。
本发明提供了一种包含非洲猪瘟病毒免疫原性蛋白的重组载体，利用所述重组载体可构建表达非洲猪瘟病毒免疫原性蛋白的重组乳酸杆菌，将所述表达非洲猪瘟病毒抗原蛋白p72和p54重组乳酸杆菌混合后，制备可用于预防非洲猪瘟病毒感染的口服活菌制剂。在本发明中，猪服食所述制 剂后，制剂中乳酸杆菌分泌的抗原蛋白(蛋白p72和p54混合物)黏附到机体细胞表面黏膜，并在黏膜表面形成抗原蛋白生物膜，抗原蛋白与靶细胞上的病毒结合位点结合，封闭黏膜表面的病毒受体蛋白位点，起到生态占位的作用，当环境中的活病毒入侵机体时，靶细胞上的病毒结合位点已被抗原蛋白生物膜完全封闭，病毒无法与靶细胞上的病毒结合位点结合，从而有效阻断病毒与细胞表面上的受体结合，起到预防非洲猪瘟的效果。
相较于疫苗，利用本发明创制的口服剂更加安全、有效、快速。本发明所述口服剂的安全性表现在有效成分只分泌病毒功能性蛋白，无病毒基因存在，不会导致病毒变异；有效性表现在所述口服剂的有效成分只分泌保护性抗原，作用于机体表面粘膜部位，覆盖非洲猪瘟病毒靶细胞所在的黏膜表面，病毒入侵时，其与黏膜表面细胞结合的位点被阻断及封闭，从而阻断病毒感染路径；快速性表现在乳酸杆菌表达的分泌蛋白直接抢占了病毒与靶细胞结合的受体从而阻断病毒感染，不需要免疫应答过程。
附图说明
图1为本发明中重组载体pVE5523-ASFV-p72的质粒结构图；
图2为本发明中重组载体pVE5523-ASFV-p54的质粒结构图；
图3为本发明中验证p72表达的扩增曲线；
图4为本发明中验证p54表达的扩增曲线。
具体实施方式
下面结合实施例对本发明进一步说明。
本发明提供了一种包含非洲猪瘟病毒免疫原性蛋白的重组载体，所述重组载体以乳酸杆菌表达载体pVE5523为基础载体，分别将编码非洲猪瘟病毒免疫原性蛋白的核苷酸序列克隆到所述基础载体的EcoRV和SalI酶切位点之间。
本发明所述非洲猪瘟病毒免疫原性蛋白包括中国吉林株非洲猪瘟病毒p72蛋白和p54蛋白；编码所述中国吉林株非洲猪瘟病毒p72蛋白的核苷酸序列如SEQ ID NO.1所示；编码所述中国吉林株非洲猪瘟病毒p54蛋白的核苷酸序列如SEQ ID NO.2所示。本发明将如SEQ ID NO.1所示的核苷酸序列克隆到所述基础载体的EcoRV和SalI酶切位点之间，形成 重组载体pVE5523-ASFV-p72，重组质粒结构如图1所示；将如SEQ ID NO.2所示的核苷酸序列克隆到所述基础载体的EcoRV和SalI酶切位点之间，形成重组载体pVE5523-ASFV-p54，重组质粒结构如图2所示。本发明对所述重组载体的构建方法并没有特殊限定，利用常规的重组载体构建方法即可。
本发明提供了一种表达非洲猪瘟病毒免疫原性蛋白的重组乳酸杆菌，所述重组乳酸杆菌包含所述重组载体。
本发明还提供了所述重组乳酸杆菌的构建方法，包括以下步骤：(1)分别将编码非洲猪瘟病毒p72蛋白的核苷酸序列和编码非洲猪瘟病毒p54蛋白的核苷酸序列克隆到pVE5523乳酸杆菌表达载体中，构建重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54；
(2)分别将重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54转化至乳酸杆菌感受态中，得到表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌。
本发明在进行克隆前，优选对GenBank中查询得到的中国吉林株非洲猪瘟病毒p72和p54基因序列进行优化，形成如SEQ ID NO.1和SEQ ID NO.2所示的序列后再进行所述克隆。
本发明在所述克隆时，优选对所述载体pVE5523以SalI/EcoRV进行双酶切后，同时将上述如SEQ ID NO.1和SEQ ID NO.2所示的序列经过同种酶的双酶切后，将酶切片段分别连接后，形成如图1和图2所示的重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54。
本发明优选利用电转化的方式分别将所述重组质粒转化ATCC393干酪乳酸杆菌感受态细胞，从而获得所述重组乳酸杆菌。本发明在得到所述重组乳酸杆菌后优选还包括将所述重组乳酸杆菌在MRS液体培养基中进行扩增，提取重组质粒后进行荧光定量PCR检测。本发明所述荧光定量PCR检测所使用的引物和扩增序列分别如下所示：
检测p72荧光定量PCR：
p72上游引物(SEQ ID NO.3)：AGTTCGGATGTCACAACGCTTG；
p72下游引物(SEQ ID NO.4)：TTTGCTTTGGTGCGGCTTGT；
p72扩增序列(SEQ ID NO.5)：
Figure PCTCN2020109703-appb-000001
p54荧光定量PCR：
p54上游引物(SEQ ID NO.6)：AGCCACTCCACAACCAGGTAC；
p54下游引物(SEQ ID NO.7)：GCCCTCCAGTTGCCATGATTAG；
p54扩增序列(SEQ ID NO.8)：
Figure PCTCN2020109703-appb-000002
本发明利用所述构建方法制备得到的重组乳酸杆菌，根据重组质粒的不同，而分别分泌重组非洲猪瘟病毒免疫原性蛋白p72和p54。
本发明还提供了一种预防非洲猪瘟感染的口服活菌制剂，所述口服活菌制剂的有效成分包括利用所述构建方法构建得到的表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌。
本发明所述口服活菌制剂中能分泌重组非洲猪瘟病毒免疫原性蛋白p72和p54的混合菌中，所述表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌的活菌比优选为(0.8～1.2)×10 8cfu:(0.8～1.2)×10 8cfu，更优选为1×10 8cfu:1×10 8cfu。
下面结合实施例对本发明提供的包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
非洲猪瘟病毒p72和p54基因序列的获得
非洲猪瘟病毒p54蛋白存在于病毒粒子内层囊膜，是ASFV的主要结构蛋白和强免疫原性蛋白之一，参与病毒对靶细胞的吸附与进入。根据GenBank中中国吉林株非洲猪瘟病毒p72和p54基因序列(P72：GenBank:MK189456.1；P54：GenBank:MK214679.1)，并对序列进行优化，改造后序列由南京金斯瑞生物科技有限公司全基因合成，碱基序列如SEQ ID NO.1和SEQ ID NO.2所示。
实施例2
重组表达载体pVE5523-ASFV-p72和pVE5523-ASFV-p54的获得
1材料与方法
1.1材料及来源
限制性内切酶SalI、EcoRV均购自NEB公司，Taq酶、dNTP、DNA Marker DL2000、DL15000、Agarose Gel DNA Purification Kit、Mini BEST Plasmid Purification Kit购自大连宝生物公司。克隆载体pVE5523由南京金斯瑞生物科技有限公司提供。
1.2试验方法
克隆载体pVE5523以SalI/EcoRV双酶切后的小片段，分别与经同样双酶切的p72和p54基因片段连接、电转化后提取质粒并送至南京金斯瑞生物科技有限公司进行测序验证。
2试验结果
重组质粒测序结果：重组质粒经基因测序后与插入的p72和p54基因片段进行序列比对，测序结果与预期相符，说明合成的p72和p54基因片段成功插入到乳酸杆菌载体pVE5523中，重组质粒构建成功，阳性质粒命名为pVE5523-ASFV-p72和pVE5523-ASFV-p54。
实施例3
表达非洲猪瘟病毒p72和p54基因的制备及检测
1材料与方法
1.1材料及来源
红霉素(Emr)购自拜尔迪生物技术有限公司。
1.2试验方法
目的基因在乳酸杆菌ATCC393中的电转化与抗性菌株的筛选：电转化后的乳酸杆菌ATCC393涂布于含5μg/ml红霉素的MRS固体培养板上，在30℃培养箱中培养72h，将平板上的菌落挑斑后接种到含5μg/ml红霉素的MRS液体培养基中，30℃静置培养72h；提取细菌中的质粒，采用荧光定量PCR进行鉴定，鉴定引物及扩增序列如下。
检测p72荧光定量PCR：
p72上游引物(SEQ ID NO.3)：AGTTCGGATGTCACAACGCTTG；
p72下游引物(SEQ ID NO.4)：TTTGCTTTGGTGCGGCTTGT；
p72扩增序列(SEQ ID NO.5)：
Figure PCTCN2020109703-appb-000003
p54荧光定量PCR：
p54上游引物(SEQ ID NO.6)：AGCCACTCCACAACCAGGTAC；
p54下游引物(SEQ ID NO.7)：GCCCTCCAGTTGCCATGATTAG；
p54扩增序列(SEQ ID NO.8)：
Figure PCTCN2020109703-appb-000004
2试验结果
采用荧光定量PCR对扩增的重组质粒进行检测，扩增曲线分别如图3～4所示，阳性重组质粒具有典型的扩增曲线，CT值19～22不等，ATCC393感受态对照没有扩增曲线。表明重组质粒pVE5523-ASFV-p54和pVE5523-ASFV-p72均已成功转化到了ATCC393感受态细胞中。
实施例4
重组乳酸杆菌表达系统的培养方法
将重组乳酸杆菌表达系统分别以1％接种量接种到乳酸杆菌MRS液 体培养基，35℃，72h后收获发酵液。
重组乳酸杆菌活菌计数
用平板表面散布法：
l.编号取无菌MRS固体琼脂培养皿9套，分别用记号笔标明10 -4、10 -5、10 -6。(稀释度)各3套。另取6支盛有4.5mL无菌水的试管，依次标是10 -1、10 -2、10 -3、10 -4、10 -5、10 -6。
2.稀释用lmL移液枪吸取已充分混匀的乳酸杆菌菌悬液(待测样品)0.5ml至10 -1的试管中，此即为10倍稀释。将10 -1试管置试管振荡器上振荡，使菌液充分混匀。另取一支lml移液枪插入10 -1试管中来回吹吸菌悬液三次，进一步将菌体分散、混匀，用此移液枪吸取10 -1菌液0.5mL至10 -2试管中，此即为100倍稀释，其余依次类推。
3.取样及涂板分别从10 -4、10 -5和10 -6的稀释菌悬液各取0.2ml，对号放入编好号的无菌琼脂培养基平皿中，用无菌的玻璃涂布棒将菌液均匀分散在琼脂培养基中，37℃恒温培养箱中培养。
4.计数培养48h后，取出琼脂培养基平皿并进行菌落计数，算出同一稀释度三个平板上的菌落平均数，并按下列公式进行计算：
每毫升中菌落形成单位(cfu)＝同一稀释度三次重复的平均菌落数×稀释倍数×5。
实施例5
将实施例4制备的能分泌重组p72和p54蛋白的乳酸杆菌分别稀释成0.8～1.2X10 8cfu/ml的菌液，并按1:1的体积比混合。将混合菌液以口服液的形式对SPF新西兰兔和SD大鼠给药，同时设置阴性对照组A口服同体积的生理盐水、对照组B口服同体积的MRS培养液，每组新西兰兔子和SD大鼠各4只，观察2周，24只动物均没有出现体温异常、过敏等异常不良现象。这表明本发明制备的能分泌重组p72和p54蛋白的乳酸杆菌安全、无副作用。
实施例6
安徽省太湖县某养殖场约存有300头猪，体型20～100kg不等，分为三个区，实验开始前，养殖二区已经有部分猪群发病并陆续死亡。2020 年1月23日对该养殖场开展重组乳酸杆菌制剂试验，试验周期为21天，分为3组，按5ml/头的剂量口服给药。其中对照组为养殖一区共112头猪，试验组分别为养殖二区和三区，实验开始前养殖一区和三区猪群处于健康状态下，二区在1月23日开展试验前已处于非洲猪瘟疫情爆发状态下。
试验结果如表1所示，对照组养殖一区猪数量由112到66头，死亡46头，死亡率达41.07％；养殖二区由于试验前已处在发病期，使用后数量由45减到28头，死亡17头，死亡率为37.78％；养殖三区猪群试验期间死亡1头，死亡率1.08％。整个试验阶段试验组(养殖二区和三区)共死亡18头，死亡率为13.04％。试验结果表明，重组乳酸杆菌制剂效果良好。
表1重组乳酸杆菌试验效果对比
Figure PCTCN2020109703-appb-000005
实施例7
湖南省益阳市某养殖场，2019年发生非洲猪瘟疫情，全场400多头育肥猪全部进行无害化处理，并对养殖场进行全面消毒。2020年4月份引进保育猪和育肥猪共120头，5月27日引进402头，引种前未进行非洲猪瘟病原检测，其中4月份引进的猪饲养半个月后，部分猪群出现不食、精神状态差等情况，死亡1头，对猪群进行血液随机抽样40头，检测非洲猪瘟病原，其中2头保育猪疑似非洲猪瘟，养殖户立即对疑似猪进行隔离，同时加强养殖场生物安全措施，一周内2头疑似猪死亡，后陆续出现死亡。5月21日将实施例4制备的重组乳酸杆菌制剂500ml加入到30公斤水中溶解，与25公斤饲料拌匀(100头猪一天的饲喂量)，每天分两次饲喂，每周2～3次。产品使用后，猪群整体健康状况明显改善，未出 现死亡现象。
表2重组乳酸杆菌产品猪场使用效果采集表
Figure PCTCN2020109703-appb-000006
5月份引进的402头猪全部采用实施例4方案生长良好，没有出现一头猪发病。
实施例8
浙江省金华市某猪场，2019年11月非瘟阳性。目前母猪存栏380头，育肥猪存栏约3900头，母猪和育肥猪全部使用实施例4制备的重组乳酸杆菌产品，年初至5月份前开始按10毫升每头每次口服，3天使用一次；5月17日开始，将实施例4制备的重组乳酸杆菌制剂500ml加入到30公斤水中溶解，与25公斤饲料拌匀(100头猪一天的饲喂量)，每天分两次饲喂，每周2～3次。2020年春节后，金华婺城区非瘟大爆发，到8月10为止，该猪场一切正常。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. 一种包含非洲猪瘟病毒免疫原性蛋白的重组载体，其特征在于，所述重组载体以乳酸杆菌表达载体pVE5523为基础载体，分别将编码非洲猪瘟病毒免疫原性蛋白的核苷酸序列克隆到所述基础载体的EcoRV和SalI酶切位点之间。
  2. 根据权利要求1所述重组载体，其特征在于，所述非洲猪瘟病毒免疫原性蛋白包括中国吉林株非洲猪瘟病毒p72蛋白和p54蛋白；编码所述中国吉林株非洲猪瘟病毒p72蛋白的核苷酸序列如SEQ ID NO.1所示；编码所述中国吉林株非洲猪瘟病毒p54蛋白的核苷酸序列如SEQ ID NO.2所示。
  3. 一种表达非洲猪瘟病毒免疫原性蛋白的重组乳酸杆菌，其特征在于，所述重组乳酸杆菌包含权利要求1或2所述重组载体。
  4. 权利要求3所述重组乳酸杆菌的构建方法，其特征在于，包括以下步骤：(1)分别将编码非洲猪瘟病毒p72蛋白的核苷酸序列和编码非洲猪瘟病毒p54蛋白的核苷酸序列克隆到pVE5523乳酸杆菌表达载体中，构建重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54；
    (2)分别将重组质粒pVE5523-ASFV-p72和pVE5523-ASFV-p54转化至乳酸杆菌感受态中，得到表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌。
  5. 根据权利要求4所述构建方法，其特征在于，在进行步骤(1)所述克隆前，还包括对GenBank中查询得到的中国吉林株非洲猪瘟病毒p72和p54基因序列进行优化，形成如SEQ ID NO.1和SEQ ID NO.2所示的序列后再进行所述克隆。
  6. 根据权利要求4所述构建方法，其特征在于，在步骤(2)得到所述重组乳酸杆菌后，还包括将所述重组乳酸杆菌在MRS液体培养基中进行扩繁增殖，提取重组质粒后进行荧光定量PCR检测。
  7. 根据权利要求6所述构建方法，其特征在于，所述荧光定量PCR 检测所用的引物对，分别包括：检测p72荧光定量PCR的引物包括p72上游引物和p72下游引物，所述p72上游引物的核苷酸序列如SEQ ID NO.3所示，所述p72下游引物的核苷酸序列如SEQ ID NO.4所示；
    检测p54荧光定量PCR的引物包括p54上游引物和p54下游引物，所述p54上游引物的核苷酸序列如SEQ ID NO.6所示，所述p54下游引物的核苷酸序列如SEQ ID NO.7所示。
  8. 权利要求1或2所述重组载体或权利要求3所述重组乳酸杆菌或利用权利要求4～7任一项所述构建方法得到的重组乳酸杆菌在预防和/或治疗非洲猪瘟感染中的应用。
  9. 权利要求1或2所述重组载体或权利要求3所述重组乳酸杆菌或利用权利要求4～7任一项所述构建方法得到的重组乳酸杆菌在制备预防和/或治疗非洲猪瘟感染药物中的应用。
  10. 根据权利要求9所述应用，其特征在于，所述药物包括口服活菌制剂。
  11. 一种预防非洲猪瘟感染的口服活菌制剂，其特征在于，所述口服活菌制剂的有效成分包括利用权利要求4所述构建方法构建得到的表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌。
  12. 根据权利要求1所述口服活菌制剂，其特征在于，所述表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌的活菌比为(0.8～1.2)×10 8cfu:(0.8～1.2)×10 8cfu。
  13. 根据权利要求12所述口服活菌制剂，其特征在于，所述表达非洲猪瘟病毒p72蛋白的重组乳酸杆菌和表达非洲猪瘟病毒p54蛋白的重组乳酸杆菌的活菌比为1×10 8cfu：1×10 8cfu。
  14. 一种治疗非洲猪瘟的方法，其特征在于，包括以下步骤：使动物摄入权利要求11～13任一项所述口服活菌制剂，所述摄入的量为5ml/动物。
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