WO2019242046A1 - 植物乳杆菌zn-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用 - Google Patents

植物乳杆菌zn-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用 Download PDF

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WO2019242046A1
WO2019242046A1 PCT/CN2018/094559 CN2018094559W WO2019242046A1 WO 2019242046 A1 WO2019242046 A1 WO 2019242046A1 CN 2018094559 W CN2018094559 W CN 2018094559W WO 2019242046 A1 WO2019242046 A1 WO 2019242046A1
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virus
lactobacillus plantarum
pedv
epidemic diarrhea
cells
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PCT/CN2018/094559
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French (fr)
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金梅林
杨丽
滑亚锋
康超
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华中农业大学
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Priority to JP2020546913A priority Critical patent/JP6968467B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

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  • the invention relates to the technical field of antiviral microecological preparations for livestock and poultry, in particular to the application of Lactobacillus plantarum ZN-3 in the preparation and treatment of swine epidemic diarrhea virus infection.
  • Intestinal diseases are a common problem in the modern pig industry, and diarrheal diseases are more common at the piglet stage, including bacterial diarrhea, viral diarrhea, nutritional diarrhea, and diarrhea caused by parasites. Among them, viral diarrhea occurs. The rate is high, the spread is fast, the spread is wide, the mortality is high, and the treatment effect is not obvious, mainly prevention. In particular, the swine epidemic diarrhea virus (PEDV) that has been outbreaking since 2010. Epidemic diarrhea is an acute and highly contagious disease that mainly shows clinical symptoms such as diarrhea, vomiting, and dehydration. The mortality rate in some large-scale pig farms is as high More than 80% is an important cause of enteritis and death in newborn piglets, causing significant economic losses to the breeding industry.
  • PEDV epidemic diarrhea virus
  • PEDV For disease, the general preventive effect will be better than the therapeutic effect, especially for viral diseases.
  • PEDV is no exception.
  • PEDV has strict cell tropism and host specificity.
  • antiviral drugs often have serious toxic side effects, with limited use and effectiveness. Therefore, there is a need to continuously seek effective methods to control viral diseases.
  • Probiotics have attracted widespread attention for their non-toxic, non-resistance, non-residual, antibacterial, anti-viral, growth-promoting, and green safety advantages.
  • Lactic acid probiotics have been widely used to prevent and treat human and animal gastrointestinal disorders. Lactic acid bacteria can tolerate the acid and alkali environment of the gastrointestinal tract of the body. Most of them are anaerobic or facultative anaerobic bacteria, which can colonize intestinal epithelial cells. Lactic acid bacteria can also produce lactic acid, lower the intestinal pH, and inhibit the invasion of harmful bacteria such as Salmonella and E. coli. Giang et al. (2010) added enterococcus faecium, Lactobacillus acidophilus, Pediococcus pentosaceus, and Lactobacillus plantarum micro-ecological preparations to the diet of weaned piglets.
  • probiotics can promote intestinal mucosal immunity and can be used as a preparation for the prevention and treatment of intestinal diseases, but research on the direct antiviral activity of probiotics is still in its infancy, especially for non-genetically engineered wild-type strains. There are few reports on antiviral activity. The antiviral effect of Lactobacillus plantarum on PEDV is currently not studied.
  • the present invention aims to obtain probiotics with antiviral activity against porcine epidemic diarrhea virus (PEDV) through research.
  • PEDV porcine epidemic diarrhea virus
  • the purpose of the present invention is to disclose the application of Lactobacillus plantarum ZN-3 in the preparation of a medicament for treating or preventing swine epidemic diarrhea virus infection.
  • Lactobacillus plantarum ZN-3 can enter the intestine through the digestive tract and inhibit the intestinal virus PEDV. Invasion and proliferation, effectively prevent and treat diseases caused by epidemic diarrhea virus.
  • the present invention adopts the following technical measures:
  • Lactobacillus plantarum ZN-3 in preparing medicines for treating or preventing swine epidemic diarrhea virus infection, including using Lactobacillus plantarum ZN-3 to prepare inhibitor of epidemic diarrhea virus (PEDV), or preparing epidemic diarrhea virus (PEDV) treatment or prevention of infections;
  • PDV epidemic diarrhea virus
  • PEDV epidemic diarrhea virus
  • Lactobacillus plantarum ZN-3 The above-mentioned deposit number of Lactobacillus plantarum ZN-3 is: CCTCC NO: M 2017286.
  • the Lactobacillus plantarum ZN-3 includes a Lactobacillus plantarum ZN-3 fermentation broth or a fermentation supernatant.
  • the present invention has the following advantages:
  • the probiotic bacterium Lactobacillus plantarum ZN-3 is used to infect cells against swine epidemic diarrhea virus (PEDV), and Lactobacillus plantarum as a potential antiviral microecological preparation has the following advantages compared with traditional antiviral chemical drugs:
  • the three treatments of Lactobacillus plantarum ZN-3 bacterial suspensions have a direct inhibitory effect on the virus, and the inhibition rates are 28.30%, 20.75%, and 22.64%; for the Lactobacillus plantarum ZN-3 fermentation supernatant, The fermentation supernatant was added first followed by PEDV dilution solution, and the fermentation broth and PEDV dilution solution were also used to directly inhibit the virus. The inhibition rates were 32.08% and 13.21%, respectively.
  • the Lactobacillus plantarum bacteria or the fermentation supernatant of pH 7.0 in the present invention has no toxic and side effect on the cells, and can all reduce the infection activity of PEDV virus to vero cells to a certain extent, and inhibit the invasion of cells by PEDV virus And infection, prevention and treatment of epidemic diarrhea in pigs.
  • Probiotics have been used for more than ten years. Although the clinical effect on gastrointestinal diseases has been confirmed to a certain extent, and the protection and enhancement of immune functions and antiviral effects of probiotics have received increasing attention, but The mechanism of action is unclear.
  • the present invention describes the inhibitory effect of probiotic Lactobacillus plantarum on epidemic diarrhea virus (PEDV) at the cellular level.
  • PEDV epidemic diarrhea virus
  • Figure 1 is a schematic diagram of the proliferation and lesions of PEDV virus on VERO cells
  • left panel uninfected PEDV cell control
  • right panel lesions formed by PEDV infection.
  • FIG. 2 is a schematic diagram of diseased cells and control cells in a 96-well plate TCID50 assay by PEDV;
  • left control cells
  • right diseased cells
  • FIG. 3 is a schematic diagram of PCR detection of PEDV amplified virus solution
  • lane 1 is a positive control; lanes 2, 3, and 4 are different pancreatin concentrations (0, 5, 10 ug / ml) for the proliferation of PEDV virus samples; lane 5 is a negative control; 6, 7, and 8 are 2, respectively. Samples 3 and 4 were repeated; lane 9 was Maker. 4 is a schematic diagram of the inhibitory effect of Lactobacillus plantarum on PEDV virus;
  • the upper left picture normal control cells
  • the upper right picture ZN-3 followed by PEDV dilution group
  • the lower left picture PEDV dilution followed by ZN-3 group
  • lower right picture virus control group.
  • test methods and conditions in the examples of the present invention are conventional methods. These embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited by these embodiments.
  • technical solutions described in the present invention are conventional solutions in the art; the reagents or materials are derived from commercial channels unless otherwise specified.
  • Lactobacillus plantarum ZN-3 in preparing medicine for treating or preventing swine epidemic diarrhea virus infection:
  • VERO cells were subcultured with 10% FBS in DMEM for about 48 hours;
  • Adherent cells use a dropper or pipette to aspirate the old culture solution in the culture vessel, and wash away the remaining old culture medium with PBS solution;
  • PEDV virus solution (CV777) stored at -80 ° C was inoculated into a culture flask with a single layer of Vero cells, and incubated at 37 ° C for 5 hours in a 5% CO2 constant temperature incubator;
  • the virus cell proliferation solution was repeatedly frozen and thawed 3 times, centrifuged at 4000 rpm for 10 min, and the supernatant virus solution was collected and stored at -80 ° C for later use.
  • RT-PCR detection of virus proliferation products The collected virus solution was used to amplify the M gene fragment of PEDV to verify the virus proliferation products.
  • PEDV virus is an RNA virus and needs reverse transcription PCR.
  • the reverse transcription system is as follows (20ul):
  • Reverse transcription reaction program 42 ° C, 60 minutes; 94 ° C, 5 minutes; 4 ° C, 10 minutes.
  • the gene amplification reaction system (25ul) is as follows:
  • Reaction procedure 94 ° C, 3 minutes; 94 ° C, 45 seconds, 53 ° C, 45 seconds, 72 ° C, 1 minute 30 seconds, 30 cycles; 72 ° C, 10 minutes; 16 ° C, 10 minutes.
  • TCID50 was used to determine the virus titer (TCID50 / 0.1ml) in the proliferated PEDV virus liquid. The specific steps are as follows:
  • PEDV virus solution was serially diluted 10-fold to 10 -6 with DMEM in a 2 ml sterile centrifuge tube;
  • the diluted virus solution was inoculated into a 96-well plate with a single layer of Vero cells, and each dilution was inoculated into a row of 8 wells.
  • the virus inoculation amount was 100ul per well, and the seventh and eighth rows were virus-free.
  • DMEM medium as a control, incubate at 37 ° C for 5 hours in a 5% CO2 incubator;
  • CPE cytopathic condition
  • the trypsin concentration has a certain effect on the proliferation of PEDV on VERO cells.
  • the virus titer is 10 -4.43 /0.1ml.
  • Lactobacillus plantarum ZN-3 was inoculated in a 150 ml MRS liquid culture medium, cultured at 37 ° C for 24 hours, centrifuged at 2400 rpm for 10 min, the supernatant was taken and the pH value of the supernatant was measured, and stored at 4 ° C until use. After centrifugation, the bacterial cell pellet was resuspended in sterile PBS, the concentration of the bacterial suspension was adjusted to 10 9 CFU / ml, and stored at 4 ° C for later use.
  • the other is to discard the incubation solution and wash it once with PBS, then add the cell maintenance solution to continue the culture, while setting up a blank cell control and a virus control;
  • the cytopathic condition was observed every day.
  • the CPE of the virus control cells reached more than 80%, the cell activity was measured by the MTT method.
  • Cytotoxicity (control OD 490 - treated group OD 490) / control OD 490 ⁇ 100%
  • the MTT method was used to indirectly determine the toxic effect of probiotic ZN-3 or its metabolites on VERO cells.
  • the results showed that 10 gradients of 10 7 CFU / ml, 10 8 CFU / ml, and 10 9 CFU / ml against VERO All cells have no toxic effect.
  • the three gradients of 10 7 CFU / ml, 10 8 CFU / ml, and 10 9 CFU / ml also had a certain degree of growth promotion (2% -4%); for the fermentation supernatant, the pH-treated group was not affected, regardless of washing With or without washing, the cell activity was significantly reduced (down by about 93%).
  • the fermentation supernatant was adjusted to pH 7.0, the activity of the unwashed group was not affected, and the activity of the washed group increased by 4.37%.
  • 10 8 CFU / ml and a fermentation supernatant adjusted to a pH of 7.0 were selected to determine the virus-inhibitory effect of probiotics and their metabolites.
  • MTT method indirectly detects the inhibition rate of probiotics on PEDV virus: the test is divided into 8 groups
  • Groups 1 to 2 were pretreated with probiotics or their metabolites, and then connected to the virus: the probiotics or metabolites were first inoculated into the cells for 90 minutes, washed and infected with 100TCID50 / 0.1ml of PEDV, and placed in an incubator. Absorb for 90 min, add cell maintenance solution after washing, and continue culturing.
  • Groups 3 to 4 were probiotics or their metabolites, which were simultaneously inserted into the cells: the probiotics or their metabolites were mixed with an equal volume of 100TCID50 / 0.1ml PEDV and added to a 96-well culture plate grown into a single layer for 90 minutes. The mixture was mixed and washed with PBS, and the cell maintenance solution was added to continue the culture.
  • Groups 5 to 6 were infected with virus first, and then inserted into probiotics or their metabolites: cells were infected with 100TCIC50 / 0.1ml PEDV, adsorbed in an incubator for 90 minutes, washed, and added with probiotics or their metabolites, and allowed to warm After 90min incubator, the cells were washed with PBS, and the cell maintenance solution was added to continue the culture.
  • 7 groups were virus controls: 100TCID50 / 0.1ml PEDV infected VERO cells, placed in the incubator for 90min, washed with PBS, and added cell maintenance solution to continue culture
  • Virus inhibition rate (the probiotic-treated group OD 490 - virus control OD 490) / (control cells OD 490 - virus control OD 490) ⁇ 100%
  • the treatment of PEDV virus by probiotics and their metabolites was measured in different ways. From the experimental results, it can be known that for ZN-3 bacteria suspension, all three treatment methods have inhibitory effects on the virus, and the inhibition rates are 28.30. %, 20.75%, 22.64%; For the fermentation supernatant, the fermentation supernatant was added first followed by PEDV dilution, and the fermentation broth and PEDV dilution were used to inhibit the virus. The inhibition rates were 32.08% and 13.21%, respectively. ; And adding PEDV dilution first, and then adding the fermentation supernatant had no inhibitory effect.

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Abstract

植物乳杆菌ZN-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用。该植物乳杆菌菌体或发酵上清液，均能在一定程度上降低PEDV病毒对宿主细胞的感染活性，抑制PEDV病毒入侵对细胞的感染，植物乳杆菌ZN-3菌体悬液的3种处理方式都对病毒有直接抑制作用；对于植物乳杆菌ZN-3发酵上清，先加发酵上清后加PEDV稀释液、同时加发酵液和PEDV稀释液2种方式对病毒也有直接抑制作用，抑制率分别为32.08％、13.21％。植物乳杆菌菌体或pH7.0发酵上清液，对细胞无毒副作用，可有效用于预防和治疗猪的流行性腹泻。

Description

植物乳杆菌ZN-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用 技术领域
本发明涉及畜禽抗病毒微生态制剂技术领域，特别涉及植物乳杆菌ZN-3在制备治疗及预防猪流行性腹泻病毒感染中的应用。
背景技术
肠道疾病是现代养猪业中普遍存在的问题，而腹泻类疾病在仔猪阶段比较易发，包括细菌性腹泻、病毒性腹泻，营养性腹泻、以及寄生虫引起的腹泻，其中病毒性腹泻发生率高、传播快、传播范围广、死亡率高，治疗效果不明显，主要以预防为主。特别是2010年至今不断爆发的猪流行性腹泻病毒(PEDV)，流行性腹泻是一种急性高度传染性疾病，主要表现出腹泻、呕吐、脱水等临床症状，在部分规模化猪场死亡率高达80％以上，是引起新生仔猪肠炎和死亡的重要原因，给养殖业造成重大的经济损失。
对于疾病来说，一般预防效果会好于治疗效果，特别是病毒性疾病，PEDV也不例外，PEDV具有严格的细胞趋性和宿主特异性。然而，由于病毒的遗传变异，有效的疫苗保护受到影响；抗病毒药物通常有严重的毒副作用，使用量和效果有限。因此，需要不断寻求可控制病毒性疾病的有效方法，益生菌因其无毒、无抗药性、无残留、抗菌、抗病毒、促生长、绿色安全的优点受到广泛关注。
乳酸类益生菌已被广泛用于预防及治疗人和动物胃肠道紊乱性疾病。乳酸菌能够耐受机体胃肠道酸碱环境，多为厌氧或兼性厌氧菌，能够在肠道上皮细胞定植。乳酸菌还可以产生乳酸，降低肠道内pH，抑制沙门氏菌、大肠杆菌等有害菌的入侵。Giang等(2010)在断奶仔猪日粮中添加屎肠球菌、嗜酸乳杆菌、戊糖片球菌、植物乳杆菌复合微生态制剂，与未添加空白对照组比较仔猪腹泻发生率下降。乳酸杆菌口服疫苗的作用效果也得到一些研究结果的证实，已有资料显示乳酸菌不仅有抗菌活性，还具有抗病毒活性。粘膜是许多细菌和病毒入侵感染机体的最主要途径，因此，粘膜免疫在预防这类感染中具有重要作用。通过粘膜途径的口服疫苗能够有效诱导机体潜在的体液免疫和粘膜免疫，是应对这类疾病的有效策略。研究报道(Van Niel等2002；张等2008)嗜酸乳杆菌能够作为轮状病毒疫苗的口服佐剂增强疫苗免疫效果。Kritas和Morrison(2007)也报道口服乳酸杆菌可以提高疫苗对PRRSV病毒感染的保护效率，而且可以提高已感染PRRSV猪的日增重。Wang XN等(2017)使用干酪乳杆菌作为抗PEDV的口服疫苗载体，结果表明干酪乳杆菌能够有效诱导机体的粘膜免疫、体液免疫、Th2类型细胞免疫，预防PEDV对机体的感染及发病。此外，益生菌作为口服疫苗具有使用方便、安全、无免疫应激、经济效益高等优点。这些资料均证明，益生菌 能够促进肠道黏膜免疫，可以作为预防和治疗肠道疾病的制剂，但是关于益生菌直接抗病毒活性的研究还处于起步阶段，特别是非基因工程改造的野生型菌株的抗病毒活性鲜有报道，关于植物乳杆菌对PEDV的抗病毒活性效果目前更是没有研究，本发明旨在通过研究获得对猪流行性腹泻病毒(PEDV)有抗病毒活性的益生菌。
发明内容
本发明的目的在于公开了植物乳杆菌ZN-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用，植物乳杆菌ZN-3可通过消化道进入肠道，抑制肠道病毒PEDV的入侵和增殖，有效预防和治疗流行性腹泻病毒引起的疾病。
为了达到上述目的，本发明采取以下技术措施：
植物乳杆菌ZN-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用，包括利用植物乳杆菌ZN-3制备成流行腹泻病毒(PEDV)的抑制剂，或是制备成流行腹泻病毒(PEDV)感染的治疗药物或是预防药物；
以上所述的植物乳杆菌ZN-3的保藏编号为：CCTCC NO：M 2017286。
以上所述的应用中，所述的植物乳杆菌ZN-3包括植物乳杆菌ZN-3发酵液或发酵上清液。
与现有技术相比，本发明具有以下优点：
本发明中用益生菌植物乳杆菌ZN-3进行抗猪流行性腹泻病毒(PEDV)对细胞感染，植物乳杆菌作为潜在的抗病毒微生态制剂与传统的抗病毒化学药物相比较具有如下优点：
1、无毒副作用、无残留、抗菌、抗病毒、促生长、绿色安全。
2、具有使用方便、安全、无免疫应激、经济效益高等优点
3、通过消化道进入肠道，抑制肠道病毒PEDV对细胞的入侵和增殖，有效预防和治疗流行性腹泻病毒引起的疾病。本发明中植物乳杆菌ZN-3菌体悬液的3种处理方式都对病毒有直接抑制作用，抑制率分别为28.30％、20.75％、22.64％；对于植物乳杆菌ZN-3发酵上清，先加发酵上清后加PEDV稀释液、同时加发酵液和PEDV稀释液2种方式对病毒也有直接抑制作用，抑制率分别为32.08％、13.21％。
4、本发明中的植物乳杆菌菌体或pH7.0发酵上清液，对细胞无毒副作用，且均能在一定程度上降低PEDV病毒对vero细胞的感染活性，抑制PEDV病毒对细胞的入侵和感染，预防和治疗猪的流行性腹泻。
5、益生菌的应用已经有十几年，虽然在临床上对消化道疾病的疗效已经得到一定程度的证实，而且益生菌对免疫功能的保护和增强作用及抗病毒作用也愈加受到关注，但其作用 机理尚不清楚，本发明从细胞水平阐述益生性植物乳杆菌对流行腹泻病毒(PEDV)的抑制作用。
附图说明
图1为PEDV病毒在VERO细胞上增殖及病变示意图；
其中，左图：未感染PEDV细胞对照，右图：感染PEDV形成的病变。
图2为PEDV在96孔板TCID50测定中病变细胞与对照细胞示意图；
其中，左图：对照细胞；右图：病变细胞。
图3为PEDV扩增病毒液PCR检测示意图；
其中，1号泳道为阳性对照；2、3、4号泳道为不同胰酶浓度(0、5、10ug/ml)增殖PEDV病毒样品；5号泳道为阴性对照；6、7、8为2、3、4样品重复；9号泳道为Maker。图4为植物乳杆菌对PEDV病毒的抑制作用示意图；
其中，左上图：正常对照细胞；右上图：先加ZN-3后加PEDV稀释液组；左下图：先加PEDV稀释液后加ZN-3组；右下图：病毒对照组。
具体实施方式
本发明实施例中试验方法和条件如无特别说明，均为常规方法。这些实施例仅用于说明本发明，本发明的保护范围不受这些实施例的限制。本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
植物乳杆菌ZN-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用：
(1)细胞培养：
VERO细胞用10％FBS的DMEM培养48小时左右传代；
细胞传代：传代时用0.25％的胰酶消化，约10分钟左右即可消化下来。具体步骤如下：
贴壁细胞用滴管或移液管吸去培养器皿中的旧培养液，用PBS溶液洗去残留的旧培养基；
向瓶内加入1-2ml消化液(0.25％胰酶)轻轻摇动培养瓶，使消化液流遍所有细胞表面；
在倒置显微镜下观察，当细胞收回突起变圆或细胞间隙增大后，应立即终止消化(吸除或倒掉消化液，加入少量的含血清的新鲜培养液，终止消化)；
用移液管吸取瓶内培养液，反复吹打消化好的细胞使其脱壁并分散，形成细胞悬液；
计数，分装到新培养瓶中，并补加一定量的含血清的新鲜培养液；
盖上瓶盖，适度拧紧后再稍回转，以利于CO2气体的进入，将培养瓶放回CO2培养箱。
(2)PEDV病毒增殖：
将消化好的Vero细胞接T75细胞培养瓶，放37℃5％CO2恒温培养箱培养至长出单层细胞；
将-80℃保存的PEDV病毒液(CV777)0.1ml接种到长有单层Vero细胞的培养瓶中，37℃5％CO2恒温培养箱孵育1h；
用移液器小心吸出病毒孵育液，加入10ml含2％FBS的DMEM细胞维持液，同时添加不同量的胰酶，最终浓度为0、5、10ug/ml胰酶，37℃5％CO2恒温培养箱培养48-72h；
每天观察细胞状态，80％以上细胞出现病变时收获病毒细胞增殖液；
病毒细胞增殖液反复冻融3次，4000rpm离心10min，收集上清病毒液-80℃保存备用。
病毒增殖产物RT-PCR检测：收集的病毒液扩增PEDV的M基因片段，对病毒增殖产物进行验证。PEDV病毒属于RNA病毒，需要做反转录PCR。
反转录体系如下(20ul)：
体系体积(ul)
Figure PCTCN2018094559-appb-000001
反转录反应程序：42℃，60分钟；94℃，5分钟；4℃，10分钟。
基因扩增反应体系(25ul)如下：
体系体积
Figure PCTCN2018094559-appb-000002
反应程序：94℃,3分钟；94℃，45秒、53℃，45秒、72℃，1分30秒、30个循环； 72℃，10分钟；16℃，10分钟。
PDEV病毒液毒价测定：采用TCID50测定增殖的PEDV病毒液中病毒的滴度(TCID50/0.1ml)，具体步骤如下：
将消化好的Vero细胞接96孔板，放37℃5％CO2恒温培养箱培养至长出单层细胞；
在2ml无菌离心管中用DMEM将PEDV病毒液连续10倍稀释至10 -6；
将稀释好的病毒液接种到长有单层Vero细胞的96孔板中，每一稀释度接种一纵排，共8孔，每孔接种病毒量为100ul,第7、8纵排接无病毒的DMEM培养基做对照，37℃5％CO2恒温培养箱孵育1h；
用移液器小心吸出病毒孵育液和DMEM培养基，加入100ul含2％FBS的DMEM细胞维持液，37℃5％CO2恒温培养箱培养120h；
每天观察并记录细胞病变(CPE)情况，按Reed-Muerch法计算PEDV病毒液的TCID50
Figure PCTCN2018094559-appb-000003
Figure PCTCN2018094559-appb-000004
由TCID50测定结果可知，胰酶浓度对PEDV在VERO细胞上的增殖有一定影响，在胰酶浓度5ug/ml条件下，病毒滴度最高为10 -4.43/0.1ml。
(3)益生菌(植物乳杆菌ZN-3的保藏号编为CCTCC NO：M 2017286)菌液及其代谢产物的制备：
将平板复壮的植物乳杆菌ZN-3接种于150ml MRS液体培养基中，37℃培养24h，2400rpm离心10min，取上清并检测上清液的pH值，4℃保存备用。离心后的菌体沉淀用无菌PBS重悬，调整菌悬液浓度为10 9CFU/ml，4℃保存备用。
(4)益生菌及代谢产物的细胞毒性检测：
在96孔板中接种细胞悬液，培养至长出单层细胞；
取植物乳杆菌ZN-3的菌悬液及代谢产物，菌悬液10倍梯度稀释，选取10 7CFU/ml、10 8CFU/ml、10 9CFU/ml 3个梯度，代谢产物取发酵上清原液和用无菌NaOH调节至pH7.0的发酵上清液；
不同处理各做一纵排，每孔加入100ul不同处理液，37℃，5％CO 2培养箱中孵育90分钟；
孵育后分2种处理：
一种是弃掉孵育液后直接加入细胞维持液继续培养；
另一种是弃掉孵育液用PBS洗涤一次后加入细胞维持液继续培养，同时设立空白细胞对照和病毒对照；
每天观察细胞病变情况，当病毒对照组细胞CPE达到80％以上时，用MTT法测定细胞活性。
细胞毒性＝(对照组OD 490－处理组OD 490)/对照组OD 490×100％
通过MTT法间接测定益生菌ZN-3或其代谢产物对VERO细胞的毒性作用，结果表明，10 7CFU/ml、10 8CFU/ml、10 9CFU/ml 3个梯度的菌悬液对VERO细胞均没有毒性作用，在孵育90min后弃掉孵育液，不用PBS洗涤直接加入维持液还可以在一定程度上(12％-14％)促进细胞的生长，在孵育90min弃掉孵育液并用PBS洗涤后，10 7CFU/ml、10 8CFU/ml、 10 9CFU/ml 3个梯度也有有一定程度促生长作用(2％-4％)；对于发酵上清，未调pH处理组，无论洗或不洗，细胞活性极显著下降(下降93％左右)，pH调至7.0的发酵上清，未洗涤组活性不受影响，洗涤组活性增加4.37％。基于此，在后续试验中分别选择10 8CFU/ml和pH值调至7.0的发酵上清液测定益生菌及其代谢产物的病毒抑制作用。
Figure PCTCN2018094559-appb-000005
(5)MTT法间接检测益生菌对PEDV病毒的抑制率：试验分为8组
1～2组分别用益生菌或其代谢产物预处理组，再接入病毒：益生菌或代谢产物先接种于细胞共培养90min，洗涤后以100TCID50/0.1ml的PEDV感染细胞，置培养箱中吸附90min，洗涤后加入细胞维持液，继续培养。
3～4组分别为益生菌或其代谢产物同时接入细胞：益生菌或其代谢产物与100TCID50/0.1ml PEDV等体积混合后加入长成单层的96孔培养板中，共培养90min，弃混合液并用PBS洗涤，加入细胞维持液继续培养。
5～6组分别为先加病毒感染细胞，在接入益生菌或其代谢产物：以100TCIC50/0.1ml PEDV感染细胞，培养箱中吸附90min后洗涤，在加入益生菌或其代谢产物，置温箱培养90min后PBS洗涤，加入细胞维持液继续培养。
7组为病毒对照：100TCID50/0.1ml PEDV感染VERO细胞，置温箱中吸附90min后PBS洗涤，加入细胞维持液继续培养
8组为正常细胞对照。
病毒抑制率＝(益生菌处理组OD 490－病毒对照组OD 490)/(细胞对照组OD 490－病毒对照组OD 490)×100％
益生菌及其代谢产物在细胞水平对PEDV病毒的抑制作用结果：
分别用不同方式处理测定益生菌及其代谢产物对PEDV病毒的抑制作用，从实验结果可以得知，对于ZN-3菌悬液，3种处理方式都对病毒有抑制作用，抑制率分别为28.30％、 20.75％、22.64％；对于发酵上清，先加发酵上清后加PEDV稀释液、同时加发酵液和PEDV稀释液2种方式对病毒有抑制作用，抑制率分别为32.08％、13.21％；而先加PEDV稀释液，后加发酵上清液没有抑制作用。
Figure PCTCN2018094559-appb-000006

Claims (3)

  1. 植物乳杆菌ZN-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用，所述植物乳杆菌ZN-3的保藏编号为：CCTCC NO：M 2017286。
  2. 植物乳杆菌ZN-3在制备猪流行性腹泻病毒抑制剂中的应用。
  3. 根据权利要求1所述的应用，所述的植物乳杆菌ZN-3为植物乳杆菌ZN-3的菌悬液或发酵上清液。
PCT/CN2018/094559 2018-06-19 2018-07-04 植物乳杆菌zn-3在制备治疗或预防猪流行性腹泻病毒感染的药物中的应用 WO2019242046A1 (zh)

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