CN113215071A - Recombinant lactococcus lactis for expressing rhabdovirus G protein of micropterus salmoides - Google Patents
Recombinant lactococcus lactis for expressing rhabdovirus G protein of micropterus salmoides Download PDFInfo
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Abstract
The invention discloses a recombinant lactococcus lactis for expressing a micropterus salmoides rhabdovirus G protein, which is a lactococcus lactis transformed with a pMG36e-His-G protein recombinant plasmid, and the preparation method of the recombinant lactococcus lactis for expressing the micropterus salmoides rhabdovirus G protein comprises the following specific steps: firstly, synthesizing a His-G protein sequence through a whole gene, then connecting the His-G protein sequence to a pMG36e plasmid to construct a pMG36e-His-G protein recombinant plasmid, then electrically converting the recombinant plasmid to lactococcus lactis, obtaining the positively cloned recombinant lactococcus lactis through erythromycin resistance screening and colony PCR identification screening, and finally analyzing the condition that the recombinant lactococcus lactis expresses the Rhabdoviral G protein of the micropterus salmoides by using an anti-His antibody and adopting western blotting. The invention successfully constructs the lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides, and can deliver the rhabdovirus G protein of the micropterus salmoides into the intestinal tract of a fish body due to the protection of the lactococcus lactis, thereby providing an important basis for the development of oral vaccines of the rhabdovirus of the micropterus salmoides.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to recombinant lactococcus lactis for expressing rhabdovirus G protein of micropterus salmoides.
Technical Field
The largemouth bass is an important economically cultured fish in China, has the advantages of fast growth, strong adaptability, no muscle stabbing, delicious meat quality and the like, and is deeply loved by consumers. In recent years, the breeding industry of the largemouth bass in China develops rapidly, and with the continuous development of the breeding in a large-scale and intensive direction, the water quality of the breeding environment and the breeding water area deteriorates gradually, so that diseases frequently occur, especially the outbreaks of diseases such as enteritis, gill rot disease, rhabdovirus disease and the like cause the largemouth bass breeding industry to be disastrous. The largemouth black bass rhabdovirus mainly infects largemouth black bass juvenile fish, and the death rate is extremely high. At present, the prevention and control of aquaculture diseases mainly depend on antibiotics, but the prevention and control of the antibiotics basically has no effect on virus diseases, and the abuse of the antibiotics causes the problems of bacterial drug resistance, drug residues, diffusion in the environment and the like.
The application of the vaccine for prevention and control can not only effectively control the occurrence of viral diseases, but also avoid the problem of drug resistance of pathogenic microorganisms caused by long-term use of the drug. In addition, aiming at the infection characteristics of largemouth black bass rhabdovirus, the operation difficulty of vaccine injection inoculation is higher, so oral inoculation and soaking inoculation are more ideal modes. However, at present, no report about the development of related vaccines exists for the rhabdovirus of the micropterus salmoides, and the prevention and the treatment of the rhabdovirus disease of the micropterus salmoides are severely restricted.
Disclosure of Invention
In response to the above needs, the applicant developed a recombinant lactococcus lactis expressing rhabdovirus G protein of micropterus salmoides to solve the above problems.
In order to achieve the purpose, the invention provides the following technical scheme:
a recombinant lactococcus lactis for expressing rhabdovirus G protein of micropterus salmoides is a lactococcus lactis transformed with pMG36e-His-G protein recombinant plasmid.
Furthermore, the recombinant lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides is disclosed, wherein the rhabdovirus G protein sequence of the micropterus salmoides is derived from Genebank and has the sequence number of MK 397811.2.
Further, the preparation method of the recombinant lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides comprises the following steps:
s1: constructing a recombinant plasmid;
s2: constructing recombinant lactococcus lactis;
s3: and (3) verifying the expression of the G protein by the recombinant lactococcus lactis.
Further, in the above method for preparing recombinant lactococcus lactis expressing rhabdovirus G protein of micropterus salmoides, the step S1 specifically includes the following steps:
obtaining sequence information of the Rhabdoviral G protein of micropterus salmoides from Genebank, synthesizing the Rhabdoviral G protein sequence of micropterus salmoides through whole genes, adding a His tag and Sac I and Hind III enzyme cutting sites, connecting to pMG36e recombinant plasmid, transferring into DH5 alpha competent cells, and sending to Shanghai engineering sequencing to verify whether the pMG36e-His-G protein recombinant plasmid is successfully constructed.
Further, in the above method for preparing recombinant lactococcus lactis expressing rhabdovirus G protein of micropterus salmoides, the step S2 specifically includes the following steps:
the recombinant plasmid is extracted by adopting a plasmid large-extraction kit, then the pMG36e-His-G protein expression plasmid is transformed into the lactococcus lactis in an electrotransformation mode, the electrotransformation condition is 2.0kV, the resistance is 400 omega, and the pMG36e plasmid replaces the pMG36e-His-G protein expression plasmid to serve as a negative control. Lactococcus lactis was then plated on M17 medium containing erythromycin and subjected to resistance screening.
After resistance screening, lactococcus lactis DNA is extracted through a kit, and then whether the lactococcus lactis with positive resistance contains a recombinant plasmid for verifying pMG36e-His-G protein is detected through PCR (polymerase chain reaction), so that the recombinant lactococcus lactis with positive clones is screened out.
Further, in the above method for preparing recombinant lactococcus lactis expressing rhabdovirus G protein of micropterus salmoides, the step S3 specifically includes the following steps:
selecting the positively cloned recombinant lactococcus lactis, culturing the positively cloned recombinant lactococcus lactis in an erythromycin-resistant M17 culture medium, analyzing the expression condition of the G protein by using an anti-His tag antibody through western blotting, and verifying whether the lactococcus lactis expressing the rhabdovirus G protein is successfully constructed; the specific operation is as follows:
(1) taking 60 mu L of recombinant lactococcus lactis, adding 15 mu L of 5 × Loading Buffer, uniformly mixing, boiling for 20min in a metal bath, adding into a sample Loading hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the recombinant lactococcus lactis whole-bacterial protein to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (each time for 5min), placing in mouse anti-His tag antibody (PBS dilution, 1:1000), incubating at 37 deg.C for 1h, and using irrelevant mouse antibody as control instead of anti-His tag antibody;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in goat anti-mouse IgG antibody (PBS diluted 1:10000) marked by HRP, and incubating for 1h at 37 ℃;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
Furthermore, the recombinant lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides is used for preparing oral vaccines of micropterus salmoides rhabdovirus.
Furthermore, the preparation method of the recombinant lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides is used for preparing oral vaccines of micropterus salmoides rhabdovirus.
The invention has at least the following beneficial effects: the recombinant lactococcus lactis prepared by the invention successfully expresses Rhabdoviral G protein of micropterus salmoides. Numerous studies have shown that rhabdovirus G protein is a key protein for interacting with a host, mediating rhabdovirus entry, is an important viral protein for inducing the host to produce neutralizing antibodies, and is also an important protein that can be used as an effector component of rhabdovirus subunit vaccines. The invention successfully constructs the lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides, and can deliver the rhabdovirus G protein of the micropterus salmoides into the intestinal tract of a fish body due to the protection of the lactococcus lactis, thereby providing an important basis for the development of oral vaccines of the rhabdovirus of the micropterus salmoides.
Drawings
FIG. 1 shows the expression of Micropterus micropterus G protein by recombinant lactococcus lactis in Western blotting analysis. Lane M: a protein Marker; lane 1: SDS-PAGE analysis of recombinant lactococcus lactis containing the pMG36e-His-G protein recombinant plasmid; lane 2: SDS-PAGE analysis of recombinant lactococcus lactis containing the empty plasmid pMG36 e; lane 3: immunoblotting of recombinant lactococcus lactis containing pMG36e-His-G protein recombinant plasmid and an anti-His tag antibody; lane 4: immunoblotting of recombinant lactococcus lactis containing empty pMG36e plasmid with anti-His tag antibody.
Detailed Description
The invention will be further illustrated by the following specific examples, which are given by way of illustration only and not by way of limitation, and the reagents described are commercially available without further description.
Examples
A recombinant lactococcus lactis for expressing rhabdovirus G protein of micropterus salmoides, wherein the recombinant lactococcus lactis: lactococcus lactis transformed with a recombinant plasmid pMG36e-His-G protein.
The Rhabdoviral G protein sequence of micropterus salmoides is derived from Genebank and has the sequence number of MK 397811.2.
The preparation method of the recombinant lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides comprises the following steps:
s1: and (5) constructing a recombinant plasmid.
Obtaining sequence information of the Rhabdoviral G protein of micropterus salmoides from Genebank, synthesizing the Rhabdoviral G protein sequence of micropterus salmoides through whole genes, adding a His tag and Sac I and Hind III enzyme cutting sites, connecting to pMG36e recombinant plasmid, transferring into DH5 alpha competent cells, and sending to Shanghai engineering sequencing to verify whether the pMG36e-His-G protein recombinant plasmid is successfully constructed.
S2: construction of recombinant lactococcus lactis
The recombinant plasmid is extracted by adopting a plasmid large-extraction kit, then the pMG36e-His-G protein expression plasmid is transformed into the lactococcus lactis in an electrotransformation mode, the electrotransformation condition is 2.0kV, the resistance is 400 omega, and the pMG36e plasmid replaces the pMG36e-His-G protein expression plasmid to serve as a negative control. Lactococcus lactis was then plated on M17 medium containing erythromycin and subjected to resistance screening.
After resistance screening, lactococcus lactis DNA is extracted through a kit, and then whether the lactococcus lactis with positive resistance contains a recombinant plasmid for verifying pMG36e-His-G protein is detected through PCR (polymerase chain reaction), so that the recombinant lactococcus lactis with positive clones is screened out.
S3: verification of recombinant lactococcus lactis expression G protein
Selecting the positive cloned recombinant lactococcus lactis, culturing the lactococcus lactis in an erythromycin-resistant M17 culture medium, analyzing the expression condition of the G protein by using an anti-His tag antibody through western blotting, and verifying whether the lactococcus lactis expressing the rhabdovirus G protein is successfully constructed. The specific operation is as follows:
(1) taking 60 mu L of recombinant lactococcus lactis, adding 15 mu L of 5 × Loading Buffer, uniformly mixing, boiling for 20min in a metal bath, adding into a sample Loading hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the recombinant lactococcus lactis whole-bacterial protein to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (each time for 5min), placing in mouse anti-His tag antibody (PBS dilution, 1:1000), incubating at 37 deg.C for 1h, and using irrelevant mouse antibody as control instead of anti-His tag antibody;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in goat anti-mouse IgG antibody (PBS diluted 1:10000) marked by HRP, and incubating for 1h at 37 ℃;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
As shown in the attached figure 1, Western blotting analysis shows that the recombinant lactococcus lactis prepared by the invention successfully expresses Rhabdoviral G protein of micropterus salmoides. Numerous studies have shown that rhabdovirus G protein is a key protein for interacting with a host, mediating rhabdovirus entry, is an important viral protein for inducing the host to produce neutralizing antibodies, and is also an important protein that can be used as an effector component of rhabdovirus subunit vaccines. The invention successfully constructs the lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides, and can deliver the rhabdovirus G protein of the micropterus salmoides into the intestinal tract of a fish body due to the protection of the lactococcus lactis, thereby providing an important basis for the development of oral vaccines of the rhabdovirus of the micropterus salmoides.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
Claims (8)
1. The recombinant lactococcus lactis for expressing the rhabdovirus G protein of the micropterus salmoides is characterized in that the recombinant lactococcus lactis is the lactococcus lactis transformed with pMG36e-His-G protein recombinant plasmid.
2. The recombinant lactococcus lactis bacterium capable of expressing Rhabdoviral G protein of micropterus salmoides according to claim 1, wherein the Rhabdoviral G protein sequence of micropterus salmoides is derived from Genebank and has the sequence number MK 397811.2.
3. The method of claim 1, wherein the recombinant lactococcus lactis is produced by the method comprising the steps of:
s1: constructing a recombinant plasmid;
s2: constructing recombinant lactococcus lactis;
s3: and (3) verifying the expression of the G protein by the recombinant lactococcus lactis.
4. The method according to claim 3, wherein the step S1 specifically comprises the following steps:
obtaining sequence information of the Rhabdoviral G protein of micropterus salmoides from Genebank, synthesizing the Rhabdoviral G protein sequence of micropterus salmoides through whole genes, adding a His tag and Sac I and Hind III enzyme cutting sites, connecting to pMG36e recombinant plasmid, transferring into DH5 alpha competent cells, and sending to Shanghai engineering sequencing to verify whether the pMG36e-His-G protein recombinant plasmid is successfully constructed.
5. The method according to claim 3, wherein the step S2 specifically comprises the following steps:
extracting recombinant plasmids by using a plasmid macroextraction kit, and then converting the pMG36e-His-G protein expression plasmid into lactococcus lactis in an electric conversion mode, wherein the electric conversion condition is 2.0kV, the resistance is 400 omega, and the pMG36e plasmid replaces the pMG36e-His-G protein expression plasmid to serve as a negative control; secondly, coating lactococcus lactis on an M17 culture medium containing erythromycin, and carrying out resistance screening;
after resistance screening, lactococcus lactis DNA is extracted through a kit, and then whether the lactococcus lactis with positive resistance contains a recombinant plasmid for verifying pMG36e-His-G protein is detected through PCR (polymerase chain reaction), so that the recombinant lactococcus lactis with positive clones is screened out.
6. The method according to claim 3, wherein the step S3 specifically comprises the following steps:
selecting the positively cloned recombinant lactococcus lactis, culturing the positively cloned recombinant lactococcus lactis in an erythromycin-resistant M17 culture medium, analyzing the expression condition of the G protein by using an anti-His tag antibody through western blotting, and verifying whether the lactococcus lactis expressing the rhabdovirus G protein is successfully constructed; the specific operation is as follows:
(1) taking 60 mu L of recombinant lactococcus lactis, adding 15 mu L of 5 × Loading Buffer, uniformly mixing, boiling for 20min in a metal bath, adding into a sample Loading hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the recombinant lactococcus lactis whole-bacterial protein to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (each time for 5min), placing in mouse anti-His tag antibody (PBS dilution, 1:1000), incubating at 37 deg.C for 1h, and using irrelevant mouse antibody as control instead of anti-His tag antibody;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in goat anti-mouse IgG antibody (PBS diluted 1:10000) marked by HRP, and incubating for 1h at 37 ℃;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
7. Use of the recombinant lactococcus lactis expressing a Rhabdoviral G protein of Lateolabrax micropterus according to any one of claims 1-2 for the preparation of oral vaccines for Rhabdoviral Lateolabrax micropterus.
8. The use of the recombinant lactococcus lactis expressing micropterus salmoides rhabdovirus G protein of claims 3-61 in the preparation of micropterus salmoides rhabdovirus oral vaccines.
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