Recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use

A technology for porcine epidemic diarrhea and Lactococcus lactis, which is applied in the field of molecular biology and can solve the problems of not being able to completely control the spread and occurrence of the epidemic.

Inactive Publication Date: 2013-05-01
DALIAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the traditional porcine epidemic diarrhea vaccine has been

Method used

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  • Recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use
  • Recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use
  • Recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Amplification of COE, the main protective antigen gene of PEDV S gene

[0046] Intestinal tissue samples of pigs infected with PEDV: Intestinal tissues of dead pigs were obtained in Jilin Dehui Nongjia Pig Farm by aseptic method.

[0047] Cut the intestinal tissue of the pig infected with PEDV into small pieces, add 2mL of sterilized normal saline, grind in a glass homogenizer, centrifuge at 3000rpm for 5min, take 100μL of the supernatant and add it to a 1.5mL Eppendorf tube, then add 1mL Trizol to extract total RNA, 200μL chloroform, Shake vigorously for 20 seconds, let stand for 2 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, take the supernatant, add 500 μL of isopropanol, mix well, let stand at room temperature for 5 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, discard the supernatant, and use 1 mL of 75% ethanol for precipitation. Wash twice, centrifuge at 7500 rpm for 5 min, discard the supernatant, dry the pellet in a desiccator until the ethan...

Embodiment 2

[0055] Example 2 Induced expression of PEDV COE gene in Lactococcus lactis

[0056] The positive recombinant bacteria and the empty plasmid control bacteria pNZ8149 / NZ3900 were inoculated into L-SGM17B medium. After culturing overnight at 30°C, the overnight culture was inoculated into 5mL medium at a ratio of 1:25 and cultivated at 30°C to OD 600 Reach 0.4~0.6. Use 1ng / mL nisin (Nisin) to induce 2~3h. After terminating the culture, take out 1mL samples, centrifuge at 12000r / min for 3min, wash the bacterial pellet with 200μL PBS, add 100μL of 10mg / mL lysozyme, 37℃ water bath for 30min, boiling water bath for 5min to inactivate lysozyme, centrifuge at 12000r / min for 3min , Discard the supernatant, add gel loading buffer (including DTT), mix well, boil for 10 min, centrifuge at 12000r / min for 3 min, take 10μL of the supernatant and load the sample, take the protein standard molecular weight as a reference, and perform 12% SDS-PAGE Electrophoresis analysis

[0057] See the result F...

Embodiment 3

[0058] Example 3 Identification of expression products

[0059] 1. Western-blot identification of expression products

[0060] After SDS-PAGE, use DYCE-40D transfer device to transfer the protein on the gel to the nitrocellulose membrane, 0.5~1mA / cm 2 Transfer 1h. After the transfer, porcine anti-PEDV high immune serum IgG was used as the primary antibody, HRP-labeled rabbit anti-pig IgG was used as the secondary antibody, and benzidine was used as the substrate for western blot analysis.

[0061] It is shown by Western-blot detection (see Image 6 Western blot analysis of the expressed product), the hybridization band is the same size as the expression band, about 20kDa, indicating that the recombinant protein can react with PEDV serum, which is consistent with the expected result.

[0062] 2. Immunization

[0063] Count the induced PEDV recombinant lactic acid bacteria under the microscope, and adjust the cell number to 1×10 9 Pieces / mL, and then use sterile PBS to make different mul...

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Abstract

The invention discloses a recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use. The construction method comprises the following steps of carrying out RT-PCR amplification to obtain a major protective antigen COE gene sequence of PEDV, carrying out clone sequencing, designing expression primers according to the sequencing result and expression vector characteristics, carrying out amplification of a PEDV COE coding sequence, connecting the PEDV COE coding sequence to a lactococcus lactis expression vector pNZ8149, and carrying out electrotransformation of the lactococcus lactis expression vector pNZ8149 into a lactococcus lactis NZ3900 cell to obtain the recombinant PEDV lactococcus lactis. The recombinant PEDV lactococcus lactis is induced by 1ng/mL of Nisin and through SDS-PAGE and Western blot analysis, a main s protein of the PEDV is expressed. An expressed recombinant PEDV lactococcus lactis vector oral vaccine can irritate production of humoral immunity and certain cellular immunity of mice. The recombinant PEDV lactococcus lactis has wide application prospects in drugs such as a PEDV vaccine.

Description

Technical field [0001] The invention relates to the technical field of molecular biology, in particular to a recombinant porcine epidemic diarrhea virus Lactococcus lactis, its construction method and application. Background technique [0002] Porcine epidemic diarrhea (PED) is a porcine intestinal infectious disease characterized by diarrhea, vomiting, and dehydration caused by Porcine epidemic diarrhea virus (PEDV) (Bi et al. 2012; Pan etal .2012;Song and Park 2012). Since 2010, swine epidemic diarrhea has spread throughout the country, causing piglet diarrhea and even death, causing serious losses to the pig industry (Mao Yayuan 2010). At present, the traditional porcine epidemic diarrhea vaccine cannot completely control the spread and occurrence of the epidemic. In this case, it is necessary to develop a new type of porcine epidemic diarrhea vaccine. [0003] PEDV spike (spike, S) glycoprotein carries the main B lymphocyte epitope and is the main structural protein that ind...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/40C12N15/74C07K16/10A61K39/42A61P31/14C12R1/46
Inventor 高凤山
Owner DALIAN UNIVERSITY
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