CN113896799A - Fusion protein, fusion protein vaccine, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a fusion protein, a fusion protein vaccine, a preparation method and an application thereof, wherein at least a part of antigen epitope region sequences in an amino acid residue sequence of TbpB protein are replaced by at least a part of antigen epitope region sequences of the amino acid residue sequence of the TbpA protein, and the obtained sequences are the amino acid residue sequences of the fusion protein. In order to make the antigenicity of TbpA obtain stable expression, the invention designs that TbpB is used as a bracket and a plurality of antigenic determinants of a plurality of surface regions of TbpA replace a plurality of larger variable loop region antigenic determinants of TbpB by utilizing the common beta-barrel structure characteristics of TbpA and TbpB, so as to obtain TbpA + B fusion protein.
Description
Technical Field
The invention relates to the field of transferrin receptor protein, and particularly relates to a fusion protein, and a preparation method, a vaccine and application thereof.
Background
The transferrin receptor protein is an important vaccine target of pathogenic bacteria-haemophilus parasuis in a pig body, the transferrin receptor protein comprises transferrin receptor protein A (TbpA) and transferrin receptor protein B (TbpB), the TbpB protein is a surface membrane protein, and the TbpB protein can be expressed in a large amount through a prokaryotic cell expression system, is easy to produce and is suitable for commercial production of vaccines; the TbpA protein is a complete outer membrane protein, and a large amount of soluble expression is not easy to obtain through a prokaryotic cell expression system, so that the production cost is high, and the TbpA protein is not suitable for commercial production of vaccines.
The existing vaccine prepared by singly adopting the TbpA protein and the vaccine prepared by singly adopting the TbpB protein have the unsatisfactory protection effect on preventing the haemophilus parasuis, which is lower than the effect of simultaneously using the two proteins, but the TbpA protein is not easy to produce and has higher cost, so that a vaccine which has the similar effect to the effect of simultaneously using the two proteins, is easy to produce and has lower cost is urgently needed.
Disclosure of Invention
Therefore, the invention provides a fusion protein, a fusion protein vaccine, a preparation method and an application thereof, in order to ensure that the antigenicity of TbpA is expressed in a stable form, the TbpB is used as a bracket by utilizing the common beta-barrel structure characteristics of the TbpA and the TbpB, a plurality of antigenic determinants of a plurality of surface regions of the TbpA are used for replacing a plurality of larger variable loop region antigenic determinants of the TbpB, and the TbpA + B fusion protein is obtained.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a fusion protein, wherein at least a part of an epitope region sequence of an amino acid residue sequence of a TbpB protein is replaced with at least a part of a subsequence of the amino acid residue sequence of the TbpA protein, and the resulting sequence is the amino acid residue sequence of the fusion protein;
the length of the subsequence is not less than the length of the antigen epitope region sequence to be replaced;
the coding amino acid sequence of the TbpA protein is shown as SEQ ID NO. 1;
the coding amino acid sequence of the TbpB protein is shown as SEQ ID NO. 2.
The hydrophilicity of the epitope region can be analyzed by DNAStar Protean software, and online website ABCcred can be applied
(website: https:// webs. iiiitd. edu.in/raghava/abcpred/index. html). Prediction of the epitope region of a particular protein by hydrophilicity analysis is a common technique used by those skilled in the art.
The epitope region of the TbpA protein comprises a variable loop region and a part of an alpha spiral region.
Preferably, the subsequence includes at least one variable loop region sequence of the sequence of amino acid residues of the TbpA protein.
Preferably, the subsequence includes at least one alpha helical region sequence of the sequence of amino acid residues of the protein TbpA.
Furthermore, a first epitope region which is replaced in the amino acid residue sequence of the TbpB protein is amino acid residues from 402 th position to 409 th position, the corresponding amino acid residue sequence of the first epitope region before replacement is SEQ ID NO. 3, and the corresponding amino acid residue sequence after replacement at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
Furthermore, the substituted second antigenic epitope region position in the amino acid residue sequence of the TbpB protein is from 433 th to 440 th amino acid residues, the corresponding amino acid residue position of the second antigenic epitope region position before the substitution is SEQ ID NO. 8, and the corresponding amino acid residue sequence after the substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
Furthermore, a third epitope region which is replaced in the amino acid residue sequence of the TbpB protein is from 459 th to 466 th amino acid residues, the corresponding amino acid residue sequence of the third epitope region before replacement at least comprises SEQ ID NO 9, and the corresponding amino acid residue sequence after replacement is one of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.
Furthermore, a fourth epitope region which is replaced in the amino acid residue sequence of the TbpB protein is 484 th to 504 th amino acid residues, the corresponding amino acid residue sequence of the fourth epitope region before replacement is SEQ ID NO. 10, and the corresponding amino acid residue sequence after replacement at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
Among them, the amino acid residue sequences of the TbpA protein and the TbpB protein are the disclosures which are easily obtained by those skilled in the art, and the fusion protein of the present invention has all the amino acid residue sequences disclosed, and those skilled in the art can easily prepare the above-mentioned fusion protein, and the preparation method thereof will not be described in detail herein.
In another aspect, the invention provides a fusion protein vaccine, and the antigen of the fusion protein vaccine comprises the fusion protein.
Preferably, the fusion protein content of the fusion protein vaccine is at least 200ug/0.5 ml.
In another aspect, the present invention provides a method for preparing the fusion protein vaccine, comprising the following steps:
preparing the fusion protein as an antigen;
and (3) compounding the fusion protein and an adjuvant according to the ratio of 9: 1.
Preferably, the adjuvant is a gel adjuvant.
The invention also provides application of the fusion protein in preparation of a medicament for preventing haemophilus parasuis.
The invention has the beneficial effects that:
in order to make TbpA antigenicity obtain stable expression, the common beta-barrel structure characteristics of TbpA and TbpB are utilized, TbpB is designed as a bracket, antigenic determinants of a plurality of surface regions of TbpA are substituted for a plurality of larger variable loop region antigenic determinants of TbpB, and TbpA + B fusion protein is obtained.
Biological preservation Instructions
The Haemophilus parasuis 5-SQ strain is Haemophilus parasuis (Haemophilus parasuis)5-SQ, CCTCC NO: m2021877, deposited by the depository: china center for type culture Collection, Address: wuhan university, preservation date: 13/7/2021, accession number: CCTCC NO: m2021877, classification name: haemophilus parasuis 5-SQ Haemophilus parauis 5-SQ.
Drawings
FIG. 1 is a diagram of a TbpB space structure provided by the present invention;
FIG. 2 is a diagram of the TbpA space structure provided by the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
The spatial structure of TbpB is shown in FIG. 1, and it has two β -barrel structures (two dashed boxes in the figure), each of which is composed of 8 β -sheets and 6 variable loop regions.
The spatial structure of TbpA is shown in FIG. 2, and it has 1 β -barrel structure, which is also composed of β -sheet and variable loop regions.
In the structures of the two proteins, the variable loop region in the beta-barrel structure is used for connecting two antiparallel beta-sheets, and the variable loop regions are also important antigenic determinants of the protein, so that the variable loop region of the beta-barrel structure on the TbpA is replaced by the variable loop region of the beta-barrel structure on the TbpB, so as to realize the TbpA + B fusion protein, and the antigenicity of the two proteins can be simultaneously realized.
Hydrophilicity analysis was performed by DNAStar Protean software, and epitope prediction was performed by using the online site ABCPred (website: https:// webs. iiitd. edu.in/raghava/ABCpred/index. html), and the epitope region (epitope) of TbpA protein was found to include the variable loop region and a portion of the alpha-helical region.
Taking Haemophilus parasuis 5-SQ strain as an example, selecting TbpB protein of 5-SQ strain and TbpA protein of 5-SQ strain, replacing four fragments of the TbpA protein of 5-SQ strain with four fragments of the TbpB protein of 5-SQ strain, wherein the specific positions of replacement are shown in the following table.
An escherichia coli expression system is adopted, pET32a is used as an expression vector, a T7 promoter is used, BL21 is used as a host bacterium, IPTG is added into a bacterial culture medium to start expression, and the TbpA + B fusion protein is prepared.
50 female guinea pigs weighing 220g-250g were selected and divided into 5 groups, and the TbpA protein, TbpB protein and TbpA + B fusion protein were mixed with gel02 adjuvant at a ratio of 9:1, the final protein content of the vaccine is 200ug/0.5ml, the immunization dose is 0.5 ml/vaccine, and the negative control group is prepared by mixing sterile normal saline and gel02 adjuvant in a ratio of 9:1, performing the proportional preparation, injecting leg muscles in different points, performing immunization twice, performing second immunization 14 days after the first immunization, performing toxicity attack 28 days after the second immunization, wherein the toxicity attack dose is the lowest total lethal dose of 5-SQ haemophilus parasuis, the method is that guinea pigs are injected in abdominal cavities, the death condition of each group of guinea pigs is observed after the toxicity attack, and the survival rate is calculated 7 days after the toxicity attack;
animal experiments prove that the TbpA + B fusion protein has similar immune effect to the simultaneous use of the TbpA and the TbpB proteins. The TbpA + B fusion protein can be expressed in a large amount of soluble forms through a prokaryotic cell expression system, is suitable for commercial production of vaccines, and the cost of the TbpA + B fusion protein is inevitably lower than the cost of the simultaneous use of the TbpA protein and the TbpB protein.
As the sequences of the Haemophilus parasuis 5-SQ strain and all the Haemophilus parasuis are universal, the TbpA + B fusion protein prepared by the preparation method disclosed by the embodiment of the invention is suitable for all the Haemophilus parasuis and has universality.
The above description is only a preferred embodiment of the present invention, and any person skilled in the art may modify the present invention or modify it into an equivalent technical solution by using the technical solution described above. Therefore, any simple modifications or equivalent substitutions made in accordance with the technical solution of the present invention are within the scope of the claims of the present invention.
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Claims (11)
1. A fusion protein is characterized in that at least a part of the epitope region sequence in the amino acid residue sequence of the TbpB protein is replaced by at least a part of the subsequence of the amino acid residue sequence of the TbpA protein, and the obtained sequence is the amino acid residue sequence of the fusion protein;
the length of the subsequence is not less than the length of the antigen epitope region sequence to be replaced;
the coding amino acid sequence of the TbpA protein is shown as SEQ ID NO. 1;
the coding amino acid sequence of the TbpB protein is shown as SEQ ID NO. 2.
2. The fusion protein of claim 1, wherein the subsequence includes at least one variable loop region sequence of amino acid residue sequences of the TbpA protein.
3. The fusion protein of claim 1, wherein the subsequence includes at least one alpha helical region sequence of amino acid residue sequences of the TbpA protein.
4. The fusion protein of claim 1, wherein the first epitope region substituted in the amino acid residue sequence of the TbpB protein is amino acid residues 402 to 409, the corresponding amino acid residue sequence of the first epitope region before substitution is SEQ ID NO. 3, and the corresponding amino acid residue sequence after substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
5. The fusion protein of claim 1, wherein the substituted second epitope region of the amino acid residue sequence of the TbpB protein is from 433 th to 440 th amino acid residues, the corresponding amino acid residue sequence of the second epitope region before the substitution is SEQ ID NO. 8, and the corresponding amino acid residue sequence after the substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
6. The fusion protein of claim 1, wherein the substituted third epitope region of the amino acid residue sequence of the TbpB protein is from 459 to 466 amino acid residues, the corresponding amino acid residue sequence of the third epitope region before substitution at least comprises SEQ ID NO. 9, and the corresponding amino acid residue sequence after substitution is one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
7. The fusion protein of claim 1, wherein the fourth epitope region substituted in the amino acid residue sequence of the TbpB protein is 484 to 504 amino acid residues, the corresponding amino acid residue sequence of the fourth epitope region before substitution is SEQ ID NO. 10, and the corresponding amino acid residue sequence after substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.
8. A fusion protein vaccine, wherein the antigen of the fusion protein vaccine comprises the fusion protein of any one of claims 1-5.
9. The fusion protein vaccine of claim 8, wherein the fusion protein content of the fusion protein vaccine is at least 200ug/0.5 ml.
10. A method of preparing the fusion protein vaccine of claim 8 or 9, comprising the steps of:
preparing the fusion protein as an antigen;
and (3) compounding the fusion protein and an adjuvant according to the ratio of 9: 1.
11. Use of the fusion protein according to any one of claims 1 to 7 for the preparation of a medicament for the prevention of haemophilus parasuis.
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| CN102869377A (en) * | 2010-03-10 | 2013-01-09 | 葛兰素史密丝克莱恩生物有限公司 | Immunogenic composition |
| CN108135992A (en) * | 2015-10-21 | 2018-06-08 | 米纳瓦克斯有限责任公司 | Immunogenic fusion proteins |
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| CN102869377A (en) * | 2010-03-10 | 2013-01-09 | 葛兰素史密丝克莱恩生物有限公司 | Immunogenic composition |
| CN103002910A (en) * | 2010-03-10 | 2013-03-27 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine composition |
| CN108135992A (en) * | 2015-10-21 | 2018-06-08 | 米纳瓦克斯有限责任公司 | Immunogenic fusion proteins |
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