CN117866858A - Recombinant lactobacillus plantarum expressing porcine rotavirus antigen and application thereof - Google Patents
Recombinant lactobacillus plantarum expressing porcine rotavirus antigen and application thereof Download PDFInfo
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- CN117866858A CN117866858A CN202311630188.3A CN202311630188A CN117866858A CN 117866858 A CN117866858 A CN 117866858A CN 202311630188 A CN202311630188 A CN 202311630188A CN 117866858 A CN117866858 A CN 117866858A
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Abstract
The application provides recombinant lactobacillus plantarum expressing porcine rotavirus antigen and application thereof. The recombinant lactobacillus plantarum contains an NSP4 gene or a VP6 gene, the nucleotide sequence of the NSP4 gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the VP6 gene is shown in SEQ ID NO. 2. The recombinant lactobacillus plantarum can effectively inhibit replication of porcine rotavirus, prevent and/or improve diarrhea caused by porcine rotavirus, improve immunity and reduce inflammation.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to recombinant lactobacillus plantarum for expressing porcine rotavirus antigen and application thereof.
Background
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Rotavirus (RV) is a double-stranded ribonucleic acid virus. Under electron microscope, rotavirus is in the shape of smooth wheel, virosome is in the shape of sphere, virus diameter is 60-80nm, it has double-layer capsid, each layer of capsid is icosahedral symmetry, core is double-strand RNA, it is made up of 11 discontinuous segments. Co-encodes (VP 1-VP 4), VP6 and VP7 together are 6 structural proteins. Studies have shown that the crystal structure of part of rotaviruses is 20 facets composed of three layers of concentric capsid proteins. The innermost capsid, which encapsulates the genome, consists of VP2 and a small amount of VP1 and VP3, which are core proteins of RV. VP6, which is the most abundant in viral proteins, constitutes the middle capsid and VP7 and VP4 constitute the outer capsid. And VP7 and VP4 are divided into G genotype and P genotype according to the outer capsid protein sequence difference. To date, 36G types and 51P types have been identified from human and various animal species. Meanwhile, the functions of each protein are preliminarily known. Such as VP1, has a profound effect on viral replication. VP3 protein has great influence on RNA replication links of viruses, and has key effects in the interaction process of viruses and host cells. VP4 and VP7 are related to virus infectivity, VP6 has a double-layer structure, so that an inner core-shell VP2 is fully contacted with outer layers VP4 and VP7 to form a trimer, the trimer is positioned in the middle layer of the virus, has a protective function on the virus, mRNA exists on the surface, is necessary for transcription, has genetic variability, and is a determining factor of species.
Rotavirus includes (NSP 1-NSP 3) and (NSP 4-NSP 6) total 6 non-structural proteins, wherein NSP1 has molecular weight of about 6.5kDa and can be used as interferon antagonistic protein. NSP2 is an octamer structure and is capable of modifying the secondary structure of RNA. NSP3 has a molecular weight of about 35kDa and can enhance viral translation. NSP4 has a molecular weight of about 27kDa and may be involved in viral replication processes and cause diarrhea as enterotoxin. In recent years, the research on the role between viruses and the host innate immune system is more and more intensive, and a rotavirus reverse genetic system based on whole plasmids is successfully established, thereby providing convenience for researching the functions of rotavirus proteins in a whole virus background.
Disclosure of Invention
It is therefore an object of the present application to provide a recombinant lactobacillus plantarum expressing porcine rotavirus antigen and uses thereof. The recombinant lactobacillus plantarum contains an NSP4 gene or a VP6 gene, the nucleotide sequence of the NSP4 gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the VP6 gene is shown in SEQ ID NO. 2. The recombinant lactobacillus plantarum can effectively inhibit replication of porcine rotavirus, prevent and/or improve diarrhea caused by porcine rotavirus, improve immunity and reduce inflammation.
Specifically, the invention provides the following technical scheme.
In a first aspect of the present invention, there is provided a recombinant lactobacillus plantarum which integrates an NSP4 gene or a VP6 gene, wherein the nucleotide sequence of the NSP4 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the VP6 gene is shown in SEQ ID No. 2.
The recombinant lactobacillus plantarum can express and produce VP6 protein or NSP4 protein, and the amino acid sequences of the recombinant lactobacillus plantarum are shown as SEQ ID NO. 3 and SEQ ID NO. 4 respectively.
The NSP4 protein is one of the non-structural proteins of rotavirus, is a multifunctional glycoprotein and is encoded by the 10 th gene. NSP4 protein plays an important role in rotavirus replication, morphogenesis and pathogenicity, and is considered the first viral enterotoxin in the world. As an enterotoxin and secretion receptor, there is an important link to the pathogenicity of viruses. NSP4 can be involved in early RV infection and affects intracellular Ca through various forms 2+ Released and causes dehydration of the body, causing diarrhea. Many studies prove that the strain is important in a disease making mechanism and plays a great role in the replication link of porcine rotavirus. Variations in NSP4 can also lead to virulence of the virus.
VP6 protein is one of structural proteins of rotavirus and is encoded by the 6 th gene. VP6 protein forms the middle capsid of rotavirus, is the most abundant and stable protein, and accounts for more than 50% of the total protein of the virus, is the core protein of the virus, and determines the group specificity of rotavirus. The VP6 protein is the primary immunogen of rotavirus, capable of inducing the generation of group-specific antibodies, for distinguishing different rotavirus groups, and involved in rotavirus replication and morphogenesis. VP6 proteins can interact with the inner and outer capsid proteins to form a stable three-layer structure and influence the transcriptional activity of the inner capsid protein. VP6 protein can also interact with non-structural proteins NSP4/NSP5, which are receptors for new bilayer particles (DLPs) in the cytoplasm, promoting the budding of DLPs from the endoplasmic reticulum. VP6 protein is involved in pathogenicity of rotavirus, and can cause apoptosis, cell cycle arrest, cytoskeletal change, cell signal transduction interference and the like through various mechanisms, so that cell dysfunction and tissue damage are caused, and virus proliferation is promoted.
DCpep (Dendritic Cells-targeting Peptide) is a peptide fragment that targets Dendritic Cells (DCs). Dendritic cells are important cells in the immune system and they play a critical role in antigen presentation and initiation of immune responses. DCpep is typically linked to a specific antigen gene to form a recombinant DNA or protein to make the gene or protein of interest. This binding helps to present the antigen directly to the dendritic cells, thereby activating an immune response.
In the embodiments of the present invention, when the inventors tried to tandem NSP4 gene fragment or VP6 gene fragment with DCpep gene, problems of slow growth rate, poor stability and reduced protein immunogenicity of recombinant strain occurred. This is probably due to the fact that the tandem of DCpep gene fragments increases the overall length and complexity of the genetic construct, resulting in instability of the expression vector and increased metabolic burden of the recombinant bacteria. In addition, such tandem may interfere with the proper folding of the NSP4 or VP6 proteins, affecting their epitope exposure, thereby reducing their activity and efficiency. Improper folding of the protein may also lead to recognition and breakdown of the protein by cellular degradation systems, further reducing the amount of protein expressed.
In some embodiments of the invention, the NSP4 gene or VP6 gene may further comprise a promoter, a terminator and a cleavage site gene, such as Xba I (TCTAGA) and Hind III (AAGCTT).
In some embodiments of the invention, the recombinant lactobacillus plantarum takes alanine racemase gene-defective lactobacillus plantarum NC 8/delta alr as an original starting strain, and the recombinant lactobacillus plantarum is NC8-pSIP409-pgsA '-VP6 or NC8-pSIP409-pgsA' -NSP4.
In some embodiments of the invention, the inventors have also tried other host strains, such as lactococcus lactis, etc., and as a result found that recombinant lactobacillus plantarum has better growth capacity, expression capacity and better tolerance to environmental stress, such as resistance to the acidic environment of the gastrointestinal tract and bile salts.
In a second aspect of the invention there is provided a composition comprising the recombinant lactobacillus plantarum described in the first aspect above.
In some embodiments of the invention, the composition contains at least one of recombinant lactobacillus plantarum NC8-pSIP409-pgsA '-VP6 and NC8-pSIP409-pgsA' -NSP 4. In a more preferred embodiment, the composition contains both recombinant Lactobacillus plantarum NC8-pSIP409-pgsA '-VP6 and NC8-pSIP409-pgsA' -NSP 4.
In a third aspect of the invention there is provided the use of a recombinant lactobacillus plantarum as described in the first aspect above or a composition as described in the second aspect above in the manufacture of a product against porcine rotavirus.
In a fourth aspect of the invention there is provided the use of a recombinant lactobacillus plantarum as described in the first aspect of the invention or a composition as described in the second aspect of the invention for the manufacture of a product for the prevention and/or amelioration of porcine rotavirus diarrhea.
In a fifth aspect of the invention there is provided the use of a recombinant lactobacillus plantarum as described in the first aspect of the invention or a composition as described in the second aspect of the invention for the preparation of a product for reducing inflammation and/or enhancing immunity in the body.
In a sixth aspect of the invention there is provided the use of a recombinant lactobacillus plantarum as described in the above first aspect of the invention or a composition as described in the above second aspect of the invention for the preparation of a product having at least one of the following functions:
(a) Increasing the content of gamma interferon (IFN-gamma) in serum;
(b) Reducing the content of interleukin 1 beta (IL-1 beta) in serum;
(c) Increasing the content of tumor necrosis factor alpha (IFN-alpha) in serum;
(d) Increasing the content of interleukin 6 (IL-6) in serum;
(e) Increasing the content of intestinal endocrine immunoglobulin a (SIgA); and
(f) Stimulating the production of specific cytokines, T cells, B cells of the spleen and/or intestinal tract;
wherein the specific cytokine comprises at least CD8+ IFN-gamma+.
In embodiments of the present invention, the products described in the above aspects may be pharmaceutical products, feeds, probiotics, scientific research reagents, etc.
In some embodiments of the invention, the feed of the invention may further comprise edible substances required for animal growth, such as foodstuffs, lipids, amino acids, trace elements, feed additives, vitamins, and the like. And, the pharmaceutical product of the present invention may comprise at least one pharmaceutical carrier or pharmaceutically acceptable adjuvant, or other therapeutically effective agent. Suitable pharmaceutical excipients may be of the type known in the art, such as solvents, buffers, diluents, etc., and may be described, for example, in the pharmaceutical excipients handbook (Handbook of Parmaceutical Excipients) by the author Paul J Shrekey et al. And the microbial ecological agent can be the bacterial powder of the recombinant lactobacillus plantarum, such as the recombinant lactobacillus plantarum obtained by culturing and fermenting the recombinant lactobacillus plantarum, and freeze-drying the obtained fermentation liquid by adding a freeze-drying protective agent (such as common freeze-drying protective agents for microorganisms, such as skimmed milk powder and the like).
In a seventh aspect of the invention, there is provided a method comprising administering to a subject a recombinant lactobacillus plantarum as described in the first aspect of the invention above or a composition as described in the second aspect of the invention above to achieve at least one of the following functions:
(1) Anti-porcine rotavirus;
(2) Preventing and/or improving porcine rotavirus diarrhea;
(3) Reducing inflammation;
(4) Enhancing immunity.
In some embodiments of the invention, the method further comprises administering to the subject a recombinant lactobacillus plantarum described in the first aspect of the invention above or a composition described in the second aspect of the invention above to achieve the function described in at least one of:
(a) Increasing the content of gamma interferon (IFN-gamma) in serum;
(b) Reducing the content of interleukin 1 beta (IL-1 beta) in serum;
(c) Increasing the content of tumor necrosis factor alpha (IFN-alpha) in serum;
(d) Increasing the content of interleukin 6 (IL-6) in serum;
(e) Increasing the content of intestinal endocrine immunoglobulin a (SIgA); and
(f) Stimulating the production of specific cytokines, T cells, B cells of the spleen and/or intestinal tract;
wherein the specific cytokine comprises at least CD8+ IFN-gamma+.
In the present invention, the term "subject" refers to an animal, such as a pig, in particular a piglet, that has been or will be the subject of treatment, observation or experiment. The methods described herein may be used for veterinary applications.
In the present invention, the effective amount required to achieve the above-described functions can be routinely determined by those skilled in the art according to conventional technical means, such as by determining or exhibiting the amount in vivo, in vitro or ex vivo experiments, and then applying the amount to practice the method of the present invention. For example, in some embodiments of the invention, the strain of the invention is administered at a dose of 1X 10 for gastric lavage 9 CFU/mL。
Advantages of the present invention compared to the prior art include:
the recombinant lactobacillus plantarum contains NSP4 genes or VP6 genes, and protein produced by expression of the recombinant lactobacillus plantarum has good immunogenicity, can successfully induce immune response of a body, induces response of T lymphocytes, successfully activates response of B lymphocytes, stimulates secretion of immunoglobulin A, improves content of gamma interferon, tumor necrosis factor alpha and interleukin 6 in serum, reduces content of interleukin 1 beta in serum, can reduce inflammation and enhance immunity of the body, and is particularly important in that the recombinant lactobacillus plantarum provided by the invention can effectively inhibit replication of porcine rotavirus, particularly can obviously reduce load of the porcine rotavirus in intestinal tracts of piglets, and can improve diarrhea caused by the porcine rotavirus and reduce intestinal damage caused by the porcine rotavirus.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: the pLP1261 vector contained the asd-alr fragment of interest PCR result (A), the plasmid pSIP409-pgsA '-VP6 fragment of interest PCR result (B) and the plasmid pSIP409-pgsA' -NSP4 fragment of interest PCR result (C).
Fig. 2: plasmid pSIP409-pgsA' -VP6 (A) map and enzyme digestion identification result.
Fig. 3: plasmid pSIP409-pgsA' -NSP4 (A) map and enzyme digestion identification result.
Fig. 4: the result of PCR of the desired fragment of plasmid pSIP409-pgsA ' -VP6 (A), the result of PCR of the desired fragment of plasmid pSIP409-pgsA ' -NSP4 (A), the result of PCR of the desired fragment of plasmid NC8-pSIP409-pgsA ' -VP6 (A), the result of PCR of the desired fragment of plasmid NC8-pSIP409-pgsA ' -NSP4 (C) and the result of PCR of the desired fragment of plasmid NC8-pSIP409-pgsA ' -NSP4 (A).
Fig. 5: western blot detects the expression results of VP6 protein (A) and NSP4 (B), wherein M: protein marker;1: empty carrier; 2 in A: NC8/pSIP409-pgsA' -VP6 (A) strain; 2 in B: NC8/pSIP409-pgsA' -NSP4 (A) strain.
Fig. 6: and the daily weight increase of piglets before and after toxicity attack.
Fig. 7: effect of each group on piglet feed/meat ratio (a) and individual group of piglet faeces evaluation results (B).
Fig. 8: content of INF-gamma in each group of piglets.
Fig. 9: content of TNF- α in each group of piglets.
Fig. 10: content of interleukin-1 beta in each group of piglets.
Fig. 11: interleukin-6 content of each group of piglets.
Fig. 12: content of SIgA in each group of piglets.
Fig. 13: and (5) quantifying the result of the duodenum after toxin attack.
Fig. 14: jejunum quantification after toxin counteracting.
Fig. 15: cecum quantification after toxin attack.
Fig. 16: b cell content in the intestinal lamina propria of piglets of each group.
Fig. 17: cd8+ IFN- γt cell content in the spleens of each group of piglets.
Fig. 18: the duodenum HE stained paraffin pathological section.
Detailed Description
The present application is further illustrated below in conjunction with specific embodiments. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in this application are all commercially available in conventional manners, and unless specifically indicated otherwise, are all used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred methods and materials described herein are presented for illustrative purposes only.
Example 1Construction of novel functional strains and protein expression
1.1 materials and methods
1.1.1 materials
1.1.1.1 Strain and plasmid Material
pSIP409-pgsA' plasmid and E.coli DH 5. Alpha. Were stored by the laboratory.
1.1.1.2 Main test Agents
The DNA plasmid miniprep kit is purchased from Guangzhou fly bioengineering Co., ltd; kanamycin sulfate and erythromycin; DL2000, 1000marker, restriction enzymes XbaI, hindIII, prime STAR Max Premix (2×) Yu Bao bioengineering limited (da); plasmid kits Fast Pure Plasmid Mini Kit, 180kDa Prestained Protein Marker were purchased from Vazyme company, SDS-PAGE loading buffer from Beijing kang as century biotechnology Co., ltd; electric stun cup (Bio-Rad Co.); sppIP is stored by Jilin province animal microecological preparation engineering research center; beef powder, yeast extract, tryptone were purchased from OXOID company, uk; DAP, D-alanine was purchased from Sigma; super GelBlue fluorescent nucleic acid gel staining reagent (EVERBRIGHT Co., U.S.A.); PBS phosphate buffer was purchased from Solarbio company.
1.1.1.3 test Main instrumentation
Gradient PCR instrument (Eppendorf AG), pipettor (Eppendorf company, germany); ultraviolet analyzer JY02S (Jun Yi electrophoresis); a chilled metal bath (TANGEN); electroporation System Gene Pμ Lser Xcell Total System #1652660 (BIO-RAD Co., U.S.A.); shaking table (Shanghai STIK Co.); L5S uv-vis spectrophotometer (Shanghai instrument electroanalysis instruments limited); full-automatic autoclave GR85DA (Sigma, germany); chemiluminescent imaging system Amersham Imager 600 (GE); -80 ℃ ultra low temperature refrigerator DW-86L626 (halr); biochemical incubator HERACELL 240i; a low temperature Centrifuge at 4 ℃ (centrafuge 5427R); electrophoresis apparatus (04DBR 02682); AI600 (Amersham Imager 600 RGB) chemiluminescent imaging system (GE); gel imaging analysis system (Universal Hood ii); flow cytometer lsrfortessa (BD company, usa).
1.1.2 Experimental methods
1.1.2.1 cloning and validation of recombinant plasmids
1.1.2.1.1 preparation of the competent preparation of coll χ6212
(1) Coli χ6212 was streaked on LB solid medium plates with DAP added and incubated overnight in a 37℃incubator.
(2) The single colony formed in the next day was selected and placed in 100mL of LB liquid medium (DAP was added), and cultured in an incubator at 37℃to measure OD600 every one hour, and when the OD600 value was about 1.0, the bacterial liquid in the incubator was taken out.
(3) And pre-cooling the bacterial liquid taken out of the incubator and a 50mL centrifuge tube in an ice box for 8min, then mixing the bacterial liquid and pouring the bacterial liquid into the centrifuge tube, and when the centrifuge is pre-cooled to 4 ℃. Centrifuge tubes were placed in axisymmetrically in a centrifuge, which was operated at 4000rpm for 8 minutes.
(4) After the work is finished, the supernatant is slowly discarded, and the PBS solution is added to be added to 45mL, and the precipitant bacteria are suspended up and down by a gun. After this time, the mixture was placed in a centrifuge, which was operated at 4000rpm for 8 minutes at 4 ℃.
(5) The supernatant was discarded again, and 45mL of 4% glycerol, prepared in advance, was added and the pellet was suspended up and down with a gun. After this time, the mixture was placed in a centrifuge, which was operated at 4000rpm for 8 minutes at 4 ℃.
(6) Repeating the step (5).
(7) The supernatant was discarded, 1.5mL of 4% glycerol was added to each 50mL centrifuge tube, and the mixture was repeatedly stirred and mixed.
(8) Respectively filling into 2mL centrifuge tubes, respectively, packaging 400 mu L of bacterial liquid into 1.5mL centrifuge tubes again, rapidly placing into liquid nitrogen, and storing in a refrigerator at-80 ℃.
1.1.2.1.2 obtaining linear DNA required for IN-FUSION
The erythromycin drug resistance genes ermL in the synthesized pSIP409-pgsA '-VP6 and pSIP409-pgsA' -NSP4 plasmids are replaced by asd and alr genes, positive clones are screened by supplementing the corresponding deletion genes of host bacteria, and drug resistance gene drift is avoided.
Primers were designed based on pSIP409-pgsA' -EGFP (A) plasmid frame, and a portion of plasmid frame linear DNA including the asd-alr gene was obtained by PCR. The primers and PCR method were as follows:
TABLE 1 asd-alr containing frame primers
TABLE 2 PCR System
TABLE 3 PCR amplification procedure
The universal primer was designed based on pSIP409-pgsA' -EGFP vector frame, and the linear DNA comprising the target gene and another part of the vector frame was obtained by PCR, which had the homology arm required for IN-FUSION with the PCR product obtained IN the previous step. The primers and PCR method are as follows, with the underlined parts being homology arms:
TABLE 4 frame primers containing the genes of interest
TABLE 5 PCR System primers
TABLE 6 PCR amplification procedure
1.1.2.1.3 cloning and screening recombinant plasmid
The framework fragments containing the target genes VP6 and NSP4 and the framework fragments containing asd-alr were cloned by IN-FUSION to obtain recombinant plasmids, and the IN-FUSION reaction system was as follows.
TABLE 7 IN-FUSION reaction System
The ligation products were transferred separately to E.coli χ6212 competence by electrotransformation.
(1) And placing the competence stored in the refrigerator with the temperature of minus 80 ℃ on ice for melting, sucking 3 mu L of connecting product into the competence after melting, flicking and mixing the connecting product uniformly by fingertips, transferring the mixture into an electric shock cup with the interval of 0.2cm, and placing the electric shock cup into an ice box for 10min after the electric shock cup is used and needs to be sterilized by ultraviolet irradiation in advance.
(2) Then the liquid outside the cup is wiped by using a mirror, and the liquid is put into an electric shock perforator to adjust the conditions to 2500V, 200 omega and 25 mu F for electric shock.
(3) After the motor cup is finished, the motor cup is placed on ice for 5min.
(4) 600 mu L of LB liquid medium is added into a electric shock cup, and after being mixed evenly, the mixture is poured into a centrifuge tube and is subjected to shaking culture for 1h at a constant temperature of 37 ℃.
(5) After that, the supernatant was separated from the precipitate by using a centrifuge at 4000rpm for 5 minutes, 200. Mu.L of the supernatant was sucked into the precipitate and stirred and mixed well.
(6) 100. Mu.L of the mixed bacterial liquid was sucked up, and an LB solid medium plate was dropped into the super clean bench, followed by plating, and the plate was placed in a incubator at 37℃overnight for culturing until single colony formation could be observed.
(7) The formed single colony is picked up and cultured in a 5mL LB liquid culture medium test tube at a constant temperature of 37 ℃ for ten hours, white turbidity precipitation is visible, and the single colony is taken out and kept in a refrigerator at-80 ℃.
Identification of 1.1.2.1.4 Positive clones
And verifying whether the sizes of target genes in the recombinant plasmid meet the expectations or not by a double enzyme digestion and PCR mode, and respectively designing primers for the two target genes for identification. Restriction enzyme identification of positive clones: the enzyme digestion system comprises the following steps:
table 8 enzyme digestion System
Table 9 enzyme digestion System
A total of 10. Mu.L of the system was placed in pcr tubes, the metal bath was adjusted to 37℃and incubated for a total of 2 hours, and 1. Mu.L of 10 XLoading Buffer was added thereto and detected by electrophoresis on a 1% agarose gel 120V for 20 min.
PCR identification of positive clones: the primers, PCR system and procedure required for PCR were as follows:
table 10 recombinant plasmid identification primers
TABLE 11 PCR identification System of pSIP409-pgsA' -Vp6 (A)
Table 12 pSIP409-pgsA' -Vp6 (A) identification PCR amplification procedure
TABLE 13 PCR identification System of pSIP409-pgsA' -NSp4 (A)
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TABLE 14 pSIP409-pgsA' -NSP4 (A) identification PCR amplification procedure
After the PCR was completed, nucleic acid electrophoresis was performed on a 1% agarose gel at 120V for 20 min.
Sequencing plasmids: after the plasmids were identified correctly, they were sent to Jilin Kumei Biotechnology Co.Ltd for sequencing.
Preparation and verification of 1.1.2.2 novel functional lactobacillus plantarum
1.1.2.2.1 preparation of NC 8/Deltaalr competent
(1) Activating strains: adding 50 mu L of NC 8/Deltaalr frozen stock solution into 5mL of MRS liquid culture medium (containing D-alanine), activating, and culturing at 37 ℃ for 12h in an anaerobic workstation;
(2) plate scribing: the inoculum was dipped with a flame sterilized loop, streaked on MRS solid medium (containing D-alanine) and sealed and incubated overnight in an anaerobic workstation at 37 ℃.
(3) Picking single colonies: the single colony was picked and added to 5mL MRS broth (containing D-alanine and Gly), placed in a 37℃incubator and removed when OD600 was around 0.6.
(4) Subculture: d-alanine liquid and Gly liquid were added to 5mL of MRS liquid medium, and 100. Mu.L of the bacterial liquid was added thereto, and the mixture was placed in a constant temperature incubator at 37℃and taken out when OD600 was about 0.2.
(5) And (3) collecting thalli: placing the bacterial liquid into a centrifuge, and setting 5000rpm; for 10min; work was performed at 4 ℃.
(6) Cleaning bacterial liquid: removing supernatant, adding 1mLPBS liquid to suspend precipitate, placing into a centrifuge again, and setting at 5000rpm; for 10min; work was performed at 4 ℃.
(7) Repeating the step (6), washing twice, and the same steps.
(8) Mixing evenly and separating pipes: and (3) dripping 300 mu L of electric shock buffer into the centrifuged test tube, uniformly mixing and precipitating, then sucking 200 mu L of bacterial liquid into a 1.5mL centrifuge tube, putting into liquid nitrogen, and then preserving in a refrigerator at-80 ℃.
1.1.2.2.2 preparation of novel functional lactobacillus plantarum: the constructed plasmid was transferred into NC 8/Deltaalr competent cells by electrotransformation.
Verification of the target gene in 1.1.2.2.3 novel functional lactobacillus plantarum: plasmids in the novel functional lactobacillus plantarum were extracted using a plasmid kit and subjected to PCR detection. The PCR system is as follows:
TABLE 15 NC8-pSIP409-pgsA' -Vp6 (A) PCR identification System
TABLE 16 NC8-pSIP409-pgsA' -Vp6 (A) identification PCR amplification procedure
TABLE 17 PCR identification System of NC8-pSIP409-pgsA' -NSp4 (A)
TABLE 18 NC8-pSIP409-pgsA' -NSp4 (A) identification PCR amplification procedure
After the PCR was completed, nucleic acid electrophoresis was performed on a 1% agarose gel at 120V for 20 min.
Verification of 1.1.2.3 novel functional lactobacillus plantarum protein expression levels: the Western blot was used to verify whether the novel functional Lactobacillus plantarum NC8-pSIP409-pgsA '-vp6 (A) and NC8-pSIP409-pgsA' -NSP4 (A) expressed the desired protein relative to the empty vector group.
1.1.2.2.2 preparation of recombinant lactobacillus plantarum protein samples:
(1) The recombinant lactobacillus plantarum is activated, 200 mu L of bacterial liquid is taken and inoculated into 5mL of MRS liquid culture medium, and the strain is cultured overnight at 37 ℃ in an anaerobic workstation.
(2) The bacterial liquid 1mL after the culture is transferred into a 50mL centrifuge tube, anaerobic culture is carried out at 37 ℃ for the next day, bacterial liquid OD600 is needed to be measured, sppIP induction peptide is added when the bacterial liquid OD600 is about 0.3, and then the bacterial liquid is cultured to be complete.
(3) The tube containing the bacterial liquid was placed in a centrifuge, adjusted to 4000rpm,15min, 500. Mu.L of supernatant bacterial liquid was retained after the end, and 1mL of PBS solution was added to dilute the pellet.
(4) Then using an ultrasonic crusher, crushing for 30min, centrifuging again, and removing the supernatant.
(5) The precipitate was diluted by adding 1mL of PBS solution. Centrifugation was performed and 200. Mu.L of supernatant was withdrawn in 1.5mL tubes and stored in a-80℃refrigerator for further use.
(6) The white cloudy precipitate remaining after centrifugation was added ddH in proportion to 2 O and 5 XSDS PAGE Buffer was followed by stirring.
(7) Boiling in 100deg.C boiling water. The centrifuge tube with the explosion-proof clamp is clamped for 15min, and then placed in a centrifuge to be adjusted to 13000rpm for 1min. After the end, the supernatant was aspirated and stored in a-20℃refrigerator.
1.1.2.2.3Western blot verification of target protein expression
And (3) glue preparation: firstly, preparing a glue-making glass plate, dripping pure water into the glue-making glass plate for 15min to see whether the liquid level is reduced, if not, firstly preparing lower glue (separating glue), dripping into the glass plate, sealing liquid by using methanol, after thirty min, solidifying JI by cementing the lower layer, pouring the methanol, wiping the liquid level by filter paper, then preparing upper glue (concentrating glue), inserting a comb into the liquid level, standing for 40 min, and waiting for solidification of the glue.
Loading and running electrophoresis: after the gel is solidified, the comb is taken out, then the glass plate is fixed in an electrophoresis tank, 1 XSDS solution is poured into the electrophoresis tank until the gel plate is submerged, and 20 mu L of Maker are added respectively; 20 mu L of protein sample, the electrophoresis apparatus was set to 80V for 2.5h, and the proteins and Maker were dispersed.
Transferring: and placing the film into a machine, placing the machine into an ice box prepared in advance, preventing the film transferring work from being damaged by too high temperature, and then inserting a power supply into the ice to transfer the film for a period of 1h at a constant current of 300 mA.
Closing: and taking out the PVDF film after the conversion, and soaking the PVDF film in a sealing liquid for sealing for 1.5 hours.
Incubation: TBST dilutions were used at 1:2000 proportion dilution murine His tag monoclonal antibody, preparing a 15mL centrifuge tube in advance, pouring diluted solution proportionally, then soaking a PVDF film in antibody diluent, placing in a shaking table at 4 ℃ for incubation for one night, taking out the solution the next day and washing for 5min by using TBST solution, and repeatedly rinsing for 3 times. Then according to the following steps of 1: diluting TBST solution and secondary antibody (HRP marked goat anti-mouse IgG) in proportion 2000, placing into a shaker at 4 ℃ for incubation for 1.5h, rinsing with PBST solution for 5min, and rinsing repeatedly for 3 times.
Taking out the PVDF film, dripping the developing solution, repeatedly knowing that the developing solution is completely soaked on the reverse side, and putting the PVDF film into an imaging system to see the final result.
1.2 results
1.2.1 obtaining PCR amplification results of vector and target fragment: primers were designed based on the pSIP409-pgsA' -EGFP (A) plasmid frame, and a portion of the plasmid frame linear DNA including the asd-alr gene was obtained by PCR, and a 4000bp band was seen. Designing a universal primer based on the pSIP409-pgsA' -EGFP vector frame, and obtaining linear DNA containing a target gene and another part of vector frame by PCR, wherein 6626bp can be seen; 5960bp band. The results are shown in FIG. 1.
1.2.2 identification of positive clones by digestion: the sizes of the target genes in the recombinant plasmids pSIP409-pgsA '-VP6 (A) and pSIP409-pgsA' -NSp4 (A) are 1206bp and 540bp through double enzyme digestion verification, and are shown in FIG. 2 and FIG. 3.
1.2.3 PCR identification of positive clones: the sizes of the target genes in the recombinant plasmids pSIP409-pgsA '-VP6 (A) and pSIP409-pgsA' -NSp4 (A) were confirmed to be 1206bp and 540bp by PCR, as shown in A and B in FIG. 6.
1.2.4 PCR validation results after transfer of the recombinant plasmid into NC8, as shown in FIGS. 6C and D.
1.2.5 Western blot detection results of recombinant lactobacillus plantarum NC8 expression protein: protein bands of 42kDa (NC 8-pSIP409-pgsA '-VP6 strain) and 25kDa (NC 8-pSIP409-pgsA' -NSP4 strain) which are consistent with the expected sizes are respectively seen by Western blot detection, and the results are shown in FIG. 5, which shows that VP6 protein and NSP4 protein can be successfully expressed, and the amino acid sequences of the protein bands are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively.
Example 2Immunization Effect of recombinant Lactobacillus plantarum constructed in example 1
In this example, two groups of novel functional Lactobacillus plantarum including NC8-pSIP409-pgsA '-VP6 (A) and NC8-pSIP409-pgsA' -NSP4 (A) constructed in example 1 were used, and after the immunized piglets were fed, the weight, feed feeding condition and diarrhea condition of the infected piglets were observed. Blood and feces are taken after toxin expelling, and ELISA experiments are carried out. T cell and B cell subpopulation changes were detected using flow cytometry. After the analysis, the virus load of pig serum and each viscera is analyzed by RT-PCR technology, and the pathological histology observation test is carried out on each viscera of the piglets, so that the immune effect of the novel functional lactobacillus plantarum on the vaccinated piglets is comprehensively evaluated, and a theoretical basis is provided for preventing and controlling PoRV diseases.
2.1 materials and methods
2.1.1 materials
Experimental strains: 2 novel functional Lactobacillus plantarum NC8-pSIP409-pgsA '-VP6 (VP 6 in the experimental group) constructed in example 1 and NC8-pSIP409-pgsA' -NSP4 (NSP 4 in the experimental group), as well as empty vectors stored in the laboratory.
Experimental animals: healthy piglets with 18 weaned 4 weeks old negative for the PoRV antigen antibodies.
Major equipment and reagents: porcine CD3-PE-Cy7 antibody and CD4-PE antibody; 1640 cell culture broth (Hyclone); the serum of the closed mice is preserved by a laboratory; antibody dilution: 1% BSA; red cell lysate, fluor 488-labeled goat anti-rabbit IgG (h+l) (bi yun biotechnology limited); FACS solution (1000 mL cultured cells with PBS,10mL FBS,0.9g sodium azide); AXYGEN PCR STRIP TUBES (corning incorporated, usa); blocking goat serum; DAPI dye liquor; anti-quenching caplets; 4% paraformaldehyde solution; a neutral gum; sodium citrate antigen retrieval solution; PRRSV-N protein rabbit-derived mab (Gene Tex); qPCR instrument (us) ABI company; tissue embedding machine, paraffin microtome, DMi8 fluorescence inverted microscope were purchased from laika instruments, inc (germany); and (5) organizing a refiner for domestic production. Other equipment and reagents are described in example 1.
2.1.2 Experimental methods
2.1.2.1 protocol
The experimental animals were grouped in Table 19.
TABLE 19 grouping of laboratory animals
Immunization and detoxification procedure: after 3 days of piglet adaptive feeding observation, 2 strains of novel functional lactobacillus plantarum NC8-pSIP409-pgsA '-VP6 and NC8-pSIP409-pgsA' -NSP4 are activated and induced to express proteins, and mixed according to the final concentration of 1 multiplied by 10 9 CFU/mL and 10 mL/head composite feeding piglet. A total of 7 drenching immunizations were performed, each time for a total of 14 days at one day intervals. In the immunization process, the heads of the piglets and the four piglets are fixed at firstThe method comprises the steps of obliquely upwards pulling the heads of piglets, breaking open the mouths and noses of the piglets, inserting a catheter into the stomach, pushing a small amount of novel functional lactobacillus plantarum into the stomach-lavage catheter for multiple times by using a lavage device, slowly pulling out the catheter after immunization is finished, sealing the mouths and noses of the piglets, and fixing for about 1min to prevent bacterial liquid from losing. After seven immunizations were completed, at one day intervals, the toxicity test was performed as in the process of gavage recombinant lactobacillus plantarum, and 1ml of 10 was administered to each of the head and limbs of the piglets in the supine position 4 After TCID50 PoRV virus dilutions, the swine was covered with hand for one minute to avoid venom flow.
Collection and processing of 2.1.2.2 samples:
a. weight and feed conversion ratio were assessed by measuring the weight of the challenged piglets at the same time interval of one day starting from the first day of the stomach-perfusing recombinant lactobacillus plantarum, recording the onset of each feeding event per day (three times a day) and observing the clinical symptoms.
b. After the toxicity is removed, the stool forms of the piglets are observed and compared, so that the damage of the PoRV to the intestinal tracts of the piglets in each group can be compared, and the digestion performance and the growth performance of the piglets are affected.
c. On day 2 after detoxification, the feces of each experimental pig were collected, ELISA was performed, and the SIgA content in the feces was analyzed.
d. And on the third day after the toxicity attack, 10mL of blood is sacrificed and stored in a refrigerator at the temperature of minus 80 ℃, and ELISA tests are carried out subsequently to analyze the IFN-gamma, IL-1 beta, TNF-alpha and IL-6 content in serum.
e. The third day after the toxin is killed, the abdominal cavity is dissected, and the intestinal tissues (twelve, empty and blind) are taken out. The tissues were analyzed for viral load by Q-PCR technique in a-80℃refrigerator.
f. The third day after the toxin is killed, the abdominal cavity is dissected, and the duodenal tissue is taken out. The tissue sample is soaked in 4% paraformaldehyde, and then is made into tissue slices in the later stage, and pathological histology observation tests are carried out.
PoRV viral load detection of each intestinal segment after 2.1.2.3 challenge
The virus load of infected piglets after virus attack can reflect the replication degree of viruses in organisms, the infection condition of the piglets and the neutralization effect of novel functional lactobacillus plantarum on the invasion of the organisms. Therefore, in this experiment, the virus load of tissues such as the duodenum, jejunum, cecum and the like of the piglets after virus challenge was measured by using an RT-PCR method.
Single tissue sample processing and RNA extraction: each intestinal tissue was removed from the-80 ℃ refrigerator, rinsed with autoclaved PBS, weighed to about 2 grams, and thoroughly ground using a pre-cooled grinder to homogenize it. The samples were added to sterilized 1.5mL EP tubes and viral RNA was extracted from the samples according to the instructions for viral RNA extraction. After measuring the concentration, cDNA was obtained by reverse transcription. After that, the analysis is performed on the machine.
2.1.2.4 preparation of Single cell suspensions
(1) Spleen single cell suspension preparation: the spleen was completely ground, the lysate was added, mixed and centrifuged, then the supernatant was removed, phosphate solution was added, mixed and centrifuged again, the supernatant was discarded, 500. Mu.L of phosphate solution was added, and single cell suspension was obtained after mixing and counted under a microscope.
(2) Preparation of mesenteric lymph node single cell suspension: the lymph nodes were completely ground, phosphate solution was added, again mixed and centrifuged, the supernatant was discarded, 500. Mu.L of phosphate solution was added, and after mixing, a single cell suspension was obtained and counted under a microscope.
2.1.2.5 flow cytometry detection: flow antibody staining.
(1) The single cell suspensions were piped 1X 10 each 6 mu.L of antibody was added to each cell in a total volume of 200. Mu.L.
(2) Based on antibody titer, a corresponding volume of flow antibody of cell surface markers was added.
(3) After full shaking and mixing, incubating in dark; standing at 4deg.C for 25min.
(4) After labelling was completed, the flow tube was filled with pre-chilled FACS buffer 1mL,180 rpm,4℃for 5min, the supernatant was discarded and the cells were resuspended with a small amount of FACS buffer.
(5) Repeating the above steps for one time, avoiding leaving a small amount of residual antibody, and reducing nonspecific staining of the cell surface.
2.1.2.6 histopathological observation and immunofluorescent staining
Paraffin section preparation: cutting intestinal tissues with proper size, placing the intestinal tissues into a 50mL centrifuge tube filled with 4% paraformaldehyde fixing solution for 3-5 days, placing the fixed intestinal tissues into a basin filled with clear water, taking out the fixed intestinal tissues, clamping the intestinal tissues by forceps, trimming the intestinal tissues with a blade, cutting the intestinal tissues with proper size, placing the intestinal tissues into a tissue embedding box, continuously flushing the intestinal tissues for 3-5 hours by running water, and starting the procedures of dehydration, transparency, wax dipping, slicing, baking, dyeing, sealing, photographing and the like, wherein the procedures are as follows:
(1) Dehydrating: the sample-embedding cassettes were immersed in alcohol of different concentrations. The liquid in the previous step is dried before being put into a new concentration in each step.
(2) And (3) transparency: taking out the tissue in 100% alcohol II, spin-drying, immersing the tissue embedding box in 100% xylene I for 7min, and transferring to 100% xylene II for 7min.
(3) Wax dipping: the sample embedding box is soaked in paraffin I and paraffin II paraffin III which are melted in advance at 58-60 ℃ for 40min respectively.
(4) Embedding: and adding the No. 58 liquid paraffin into an embedding machine in advance, taking out the immersed liquid paraffin III after melting, putting the immersed liquid paraffin III into a small box of the embedding machine, respectively taking out the immersed intestinal tissues, putting the immersed intestinal tissues into an embedding shell, paying attention to the placing position of tissue samples, and dripping and filling the liquid paraffin until the wax liquid is cooled and solidified for subsequent treatment.
(5) Slicing: the embedding machine is placed in a ventilated, dry and stable environment. Placing the embedded intestinal tissue wax block into an ice box, fixing in a slicing machine after ice, adjusting the thickness to 2.5 mu m, slicing, spreading the cut paraffin tissue sample into a water tank heated to 42 ℃ and purified water, taking out the paraffin tissue sample quickly with a glass slide carefully, and marking the names of the tissues.
(6) Baking slices: placing the glass slide in a staining rack, baking in an incubator at 80 ℃ for about 40min, taking out, observing whether white spots exist on the glass slide, and if no spots exist, baking the glass slide.
(7) Dyeing: firstly, putting dye liquor required by dyeing into a light-proof kitchen in sequence, pouring the dye liquor into a marked dyeing box, preparing a barrel of clean purified water in advance, respectively putting a dyeing frame into xylene I and xylene II for 5min, then putting the dye frames into purified water for washing for 1min, then putting 100% alcohol for 1min, 95% alcohol for 1min, 90% alcohol for 1min, 80% alcohol for 1min and 70% alcohol for 1min, or soaking the dye frames up and down for 8 times, then putting the dye frames into purified water for washing, and then putting hematoxylin dye liquor for dyeing; the staining time is determined by the slice thickness and tissue type, and staining is often observed. Washing with water after dyeing, acidifying with hydrochloric acid for 1min, washing with water, observing whether the cell nucleus is blue or not, and re-dyeing if the cell nucleus is light. Then adding eosin dye solution for not more than 3.5min, taking out, washing with water, and soaking in 95% alcohol I and 95% alcohol II for 8 times. After dyeing is completed, the dyeing rack is put into a baking oven for about 5min until the dyeing rack is completely baked.
(8) Sealing piece: taking out the dried pieces, dripping one to two drops of neutral gum into each piece in a ventilation kitchen, sealing the pieces with a cover glass, and writing a label. The gum was left in the hood for a period of 2-3 days until the gum was completely sealed.
(9) Photographing: placing the sheet in a DMi8 fluorescence inverted microscope to find intestinal villi; bleeding points; and taking photographs of pathological tissues with different multiples of the parts such as the shedding tissues.
2.1.2.7 statistical analysis of data: and respectively counting feed-meat ratio growth performance indexes by using Excel software, using flow jo_v10.6.2 analysis software for flow cytometry, using Graphpad Prism8 software for data statistics, and using t-test method for data difference statistics analysis, wherein P <0.05 represents that the difference is more obvious, P >0.05 represents that the difference is not obvious, P < 0.01 represents that the difference is obvious, and P < 0.001 represents that the difference is extremely obvious.
2.2 results
2.2.1 novel functional Lactobacillus plantarum effects on daily gain of piglets
The daily gain of pigs is one of the production performance indexes of the pigs, and the whole-course daily weighing mode is adopted in the experiment to compare the daily gain amplitude of piglets of each experimental group before and after the toxicity attack. Fig. 6 shows that the healthy group of piglets with PBS without toxicity attack is in a continuous rising trend, and the falling amplitude of the piglets with PBS toxicity attack and the piglets with empty carrier group is quite obvious after toxicity attack. The new functional lactobacillus plantarum VP6 group and the NSP4 group have smaller weight reduction, the compound group (the new functional lactobacillus plantarum VP6 and the NSP 4) still presents small increase after toxicity attack, and the daily gain change of piglets indicates that the weight reduction symptoms caused by the PoRV can be prevented and treated to a certain extent between the new functional lactobacillus plantarum VP6 and the NSP4 and the mixture of the new functional lactobacillus plantarum VP6 and the NSP4, and particularly the effect of the compound group is most obvious, and the effect is hardly influenced before and after toxicity attack.
2.2.2 New functional Lactobacillus plantarum effects on piglet feed to meat ratio
Feed to meat ratio (FCR) refers to the amount of feed consumed by piglets which are weighted by one kilogram, and is an important indicator by which the growth performance of each group of piglets can be evaluated. If the feed-meat ratio is high, the feeding amount is more, and the growth of piglets is less. If the meat feed ratio is low, the feeding amount is small, and the piglets grow more. As can be seen from the result a in fig. 7: the meat-to-meat ratio of the PBS healthy group is 2.55, the meat-to-meat ratio of the PBS challenge group is 2.48, the meat-to-meat ratio of the empty carrier group is 2.36, the meat-to-meat ratio of the novel functional lactobacillus plantarum VP6 group is 2.10, the meat-to-meat ratio of the novel functional lactobacillus plantarum NSP4 group is 1.88, and the meat-to-meat ratio of the composite bacteria group is 1.73. The feed conversion ratio of PBS challenge group is obviously different from that of the new functional lactobacillus plantarum VP6 group (P is less than 0.01); the feed conversion ratio of PBS challenge group is obviously different from that of the new functional lactobacillus plantarum NSP4 group (P is less than 0.01); the difference in feed conversion ratio was very significant (P < 0.001) in the PBS challenge group compared to the complex group, indicating that both recombinant strains and their complexes were able to significantly reduce feed conversion ratio.
2.2.3 Effect of novel functional Lactobacillus plantarum on the morphological scoring of piglet faeces
After the toxicity is removed, the pig of each experimental group can be compared with the pig of each experimental group to cause damage to the intestinal tracts of the piglets and the influence of the flora, so that the digestion, absorption and growth performance of the piglets on nutrition can be influenced. The scoring criteria were: wet forming and fluffing to 0 minutes of normal feces, 1 minute to soft feces, mild diarrhea, dry hard to 2 minutes to thinner feces, medium diarrhea, 3 minutes to watery feces, severe diarrhea, and 0 minutes to optimal. As shown in fig. 7B, after the toxicity attack, the damage to the intestinal tracts and the bacterial colony of the piglets of the PBS toxicity attack group and the empty carrier group are seriously affected, and the new functional lactobacillus plantarum VP6 and NSP4 groups have remarkable prevention and treatment effects on diarrhea caused by the PoRV, so that the prevention and treatment effects of the composite bacterial group are optimal.
2.2.4 immune indicators INF-gamma, TNF-a, IL-1 beta, IL-6, SIgA detection results
2.2.4.1 immune index INF-gamma detection results
The results of fig. 8 show that the new functional lactobacillus plantarum NSP4 group and the compound group (VP 6 and NSP 4) are significantly different from the control group (PBS virus attacking group) on the third day after the piglet virus attacking (P < 0.05), which shows that the INF-gamma content in the blood of piglets fed with the new functional lactobacillus plantarum NSP4 group and the compound recombinant lactobacillus plantarum group (VP 6 and NSP 4) is significantly increased compared with the control group and is superior to the new functional lactobacillus plantarum VP6 group, and the new functional lactobacillus plantarum NSP4 group and the compound group (VP 6 and NSP 4) can significantly improve the antiviral ability of the piglets.
2.2.4.2 immune index TNF-alpha detection results
The results of fig. 9 show that the new functional lactobacillus plantarum VP6, the new functional lactobacillus plantarum NSP4 and the complex groups (VP 6 and NSP 4) are significantly different from the control group (PBS challenge group) (P < 0.01) on the third day after the piglet challenge, which indicates that the TNF-alpha content in the blood of piglets fed with the new functional lactobacillus plantarum VP6, the new functional lactobacillus plantarum NSP4 and the complex groups (VP 6 and NSP 4) is significantly reduced compared with the control group, indicating that the new functional lactobacillus plantarum VP6, the new functional lactobacillus plantarum NSP4 and the complex groups (VP 6 and NSP 4) can significantly improve the antiviral ability of piglets.
2.2.4.3 immune index IL-1 beta detection result
The results of fig. 10 show that the difference between the novel functional lactobacillus plantarum VP6 and the control group (PBS challenge group) is obvious (P < 0.01) on the third day after the piglet is challenged, the differences between the novel functional lactobacillus plantarum NSP4 group, the complex group (VP 6 and NSP 4) and the PBS healthy group and the control group (PBS challenge group) are extremely obvious (P < 0.001), which indicates that the IL-1 beta content in the blood of piglets fed with the novel functional lactobacillus plantarum VP6, the novel functional lactobacillus plantarum NSP4 group and the complex group (VP 6 and NSP 4) is obviously reduced compared with the control group, and the novel functional lactobacillus plantarum NSP4 group and the complex group (VP 6 and NSP 4) can obviously improve the antiviral capability of piglets and are superior to the novel functional lactobacillus plantarum VP6 group.
2.2.4.4 detection results of immune index IL-6
The third day after the piglet is challenged, the novel functional lactobacillus plantarum VP6 has no obvious difference from the control group (PBS challenged group), the novel functional lactobacillus plantarum NSP4 has obvious difference (P < 0.01) from the control group (PBS challenged group), the composite group (VP 6 and NSP 4) has obvious difference from the control group (PBS challenged group) (P < 0.05), which indicates that the IL-6 content in the blood of the piglets fed with the novel functional lactobacillus plantarum NSP4 and the composite recombinant lactobacillus plantarum group (VP 6 and NSP 4) is obviously increased compared with the control group, and the novel functional lactobacillus plantarum NSP4 can obviously improve the antiviral capability of the piglets and is superior to the composite group (VP 6 and NSP 4).
2.2.4.5 detection result of immune index SIgA
The results of fig. 12 show that the difference between the novel functional lactobacillus plantarum VP6 and the control group (PBS challenge group) is very significant (P < 0.001), the difference between the novel functional lactobacillus plantarum NSP4 group and the control group (PBS challenge group) is very significant (P < 0.001), and the difference between the compound groups (VP 6 and NSP 4) and the control group (PBS challenge group) is very significant (P < 0.001), which indicates that the SIgA content in the feces of piglets fed with the novel functional lactobacillus plantarum VP6, the novel functional lactobacillus plantarum NSP4 group and the compound groups (VP 6 and NSP 4) is very significantly increased compared with the control group, and the novel functional lactobacillus plantarum NSP4 group and the novel functional lactobacillus plantarum compound groups (VP 6 and NSP 4) can significantly improve the antiviral ability of piglets.
2.2.5 quantitative results of duodenal, jejunum, cecum viral loads after challenge
2.2.5.1 duodenal viral load quantification results
After the duodenum sample is extracted with virus nucleic acid and quantitatively analyzed (figure 13), the duodenum virus load of the novel functional lactobacillus plantarum VP6 group is lower than (P < 0.001) that of the control group (PBS virus attack group), the duodenum virus load of the novel functional lactobacillus plantarum NSP4 group is lower than (P < 0.001) that of the control group (PBS virus attack group), and the duodenum virus load of the composite (VP 6 and NSP 4) groups is lower than (P < 0.001) that of the control group (PBS virus attack group), so that the virus load of the novel functional lactobacillus plantarum VP6 group, the novel functional lactobacillus plantarum NSP4 group and the novel functional lactobacillus plantarum composite group (VP 6 and NSP 4) in the duodenum of infected piglets can be reduced.
2.2.5.2 jejunal viral load quantification results
After virus nucleic acid is extracted from the jejunum sample and quantitative analysis is carried out (figure 14), the jejunum virus load of the novel functional lactobacillus plantarum VP6 group is lower than (P < 0.001) that of a control group (PBS virus attack group), the jejunum virus load of the novel functional lactobacillus plantarum NSP4 group is lower than (P < 0.001) that of the control group (PBS virus attack group), the jejunum virus load of the novel functional lactobacillus plantarum complex (VP 6 and NSP 4) is lower than (P < 0.001) that of the control group (PBS virus attack group), and the novel functional lactobacillus plantarum VP6 group, the novel functional lactobacillus plantarum NSP4 group and the complex groups (VP 6 and NSP 4) can reduce the virus load in jejunum of infected piglets.
2.2.5.3 cecal viral load quantification results
After the cecum sample is extracted and quantitatively analyzed (figure 15) to show that the cecum virus load of the novel functional lactobacillus plantarum VP6 group is lower than (P < 0.01) that of the control group (PBS virus attack group), the cecum virus load of the novel functional lactobacillus plantarum NSP4 group is lower than (P < 0.01) that of the control group (PBS virus attack group), and the cecum virus load of the novel functional complex (VP 6 and NSP 4) groups is lower than (P < 0.01) that of the control group (PBS virus attack group), so that the novel functional lactobacillus plantarum VP6 group, the novel functional lactobacillus plantarum NSP4 group and the complex groups (VP 6 and NSP 4) can reduce the virus load in the cecum of infected piglets.
2.2.6 flow cytometry results
2.2.6.1 novel functional Lactobacillus plantarum influence on B cells
B cells are an important component of humoral immunity and intestinal mucosal immunity. The present invention investigated the level of B cell activation in the Lamina Propria (LPL) of the gut. The results in fig. 16 show that after challenge of the experimental piglets of each group, the blank group (PBS healthy group), the negative control group (empty vector group), the positive control group (PBS challenge group), the novel functional lactobacillus plantarum VP6 group, the novel functional lactobacillus plantarum NSP4 group and the novel functional lactobacillus plantarum complex group (VP 6 and NSP 4) have B cell numbers of 32.4%, 22.4%, 28.0%, 42.2%, 42.7% and 51.5% in the intestinal Lamina Propria (LPL), respectively. The novel functional lactobacillus plantarum VP6, NSP4 and the compound group in the intestinal lamina propria are extremely obvious in difference from the control group (PBS virus attack group) (P < 0.001). The results show that after virus attack, the novel functional lactobacillus plantarum VP6, NSP4 and complex can continuously activate organism to generate B cells in the intestinal lamina propria so as to achieve the protection capability. The ability of the complex to activate the body in the lamina propria of the intestine is more pronounced.
2.2.6.2 novel functional Lactobacillus plantarum influence on TH1 immune response
Since cytotoxic T cells are capable of inducing TH1 intracellular cells to mount an immune response by secreting IFN-gamma factor. The novel functional lactobacillus plantarum NSP4 and the complex can activate CD8+T cells to secrete IFN-gamma, and the effect of the complex is more obvious. The differences between the new functional Lactobacillus plantarum VP6, NSP4 and the compound group and the control group (PBS challenge) are very significant after virus attack (P < 0.01), and the results are shown in FIG. 17.
2.2.7 histopathological observations of piglets after challenge
In order to detect the protection effect of the novel functional lactobacillus plantarum on the experimental pig, the experimental pig is killed in the experiment to prepare a pathological section of the duodenum, and histopathological observation is carried out. As a result, the structure of the duodenal villi of the PBS healthy group is complete; the new functional lactobacillus plantarum VP6 group and NSP4 group have fewer duodenal villus atrophy, scattering and fracture conditions compared with a control group (PBS virus attack group) and an empty vector group; the duodenal lines are distributed sparsely, and there are occasionally muscle layer breaks. The novel functional lactobacillus plantarum complex (VP 6 and NSP 4) has complete intestinal tract structure; the intestinal lines are uniformly distributed, and no muscular layer fracture condition occurs. The novel functional lactobacillus plantarum VP6 group, the novel functional lactobacillus plantarum NSP4 group and the compound group (VP 6 and NSP 4) can obviously improve the antiviral capability of the duodenum of piglets. The antiviral ability of the complex group (VP 6 and NSP 4) was better than that of the VP6 group and NSP4 group, and the result is shown in FIG. 18.
The foregoing description is only a preferred embodiment of the present application, and is not intended to limit the present application, but although the present application has been described in detail with reference to the foregoing embodiment, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (10)
1. A recombinant lactobacillus plantarum is characterized by comprising an NSP4 gene or a VP6 gene, wherein the nucleotide sequence of the NSP4 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the VP6 gene is shown as SEQ ID NO. 2.
2. The recombinant lactobacillus plantarum of claim 1, wherein the NSP4 gene or VP6 gene may further comprise a promoter, a terminator and a cleavage site gene;
preferably, the cleavage sites are Xba I and Hind III.
3. The recombinant lactobacillus plantarum according to claim 1, characterized in that the recombinant lactobacillus plantarum takes alanine racemase gene-defective lactobacillus plantarum NC 8/deltaalr as an original starting strain, and the recombinant lactobacillus plantarum is NC8-pSIP409-pgsA '-VP6 or NC8-pSIP409-pgsA' -NSP4;
preferably, the recombinant lactobacillus plantarum expression produces VP6 protein or NSP4 protein, the amino acid sequences of which are shown in SEQ ID NO. 3 and SEQ ID NO. 4 respectively.
4. A composition comprising the recombinant lactobacillus plantarum of any of claims 1 to 3.
5. The composition of claim 4, wherein the composition comprises recombinant lactobacillus plantarum NC8-pSIP409-pgsA '-VP6 and/or NC8-pSIP409-pgsA' -NSP4.
6. Use of a recombinant lactobacillus plantarum according to any of claims 1 to 3 or a composition according to claim 4 or 5 for the preparation of a product against porcine rotavirus.
7. Use of a recombinant lactobacillus plantarum according to any of claims 1 to 3 or a composition according to claim 4 or 5 for the preparation of a product for the prevention and/or amelioration of porcine rotavirus diarrhea.
8. Use of a recombinant lactobacillus plantarum according to any of claims 1 to 3 or a composition according to claim 4 or 5 for the preparation of a product for reducing inflammation and/or enhancing the immunity of the body.
9. Use of a recombinant lactobacillus plantarum of any of claims 1 to 3 or a composition of claim 4 or 5 for the preparation of a product having at least one of the following functions:
(a) Increasing the content of gamma interferon in serum;
(b) Reducing the content of interleukin 1 beta in serum;
(c) Increasing the content of tumor necrosis factor alpha in serum;
(d) Increasing the content of interleukin 6 in serum;
(e) Increasing the content of the endocrine immunoglobulin A in the intestinal tract; and
(f) Stimulating the production of specific cytokines, T cells, B cells of the spleen and/or intestinal tract;
preferably, the specific cytokine comprises cd8+ IFN- γ+.
10. The product according to any one of claims 6 to 9, characterized in that it comprises pharmaceutical products, feeds, probiotics and scientific agents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN116875520A (en) * | 2023-07-12 | 2023-10-13 | 吉林农业大学 | Lactic acid bacteria expressing ribosome inactivating protein and application thereof in resisting rotavirus |
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| CN116875520A (en) * | 2023-07-12 | 2023-10-13 | 吉林农业大学 | Lactic acid bacteria expressing ribosome inactivating protein and application thereof in resisting rotavirus |
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