CN111304224A - Group B neisseria meningitidis recombinant pilin Fim and preparation method and application thereof - Google Patents
Group B neisseria meningitidis recombinant pilin Fim and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological pharmacy, and particularly relates to a group B neisseria meningitidis recombinant bacterial hair protein Fim, and a preparation method and application thereof. The invention provides a group B neisseria meningitidis recombinant pilin Fim gene, wherein the nucleotide sequence of the group B neisseria meningitidis recombinant pilin Fim gene is shown as SEQ ID NO. 1. The invention discovers that the recombinant protein is a key molecule for adhesion and permanent planting of the neisseria meningitidis for the first time, clones the molecule to a protein expression vector for large-scale expression, and prepares the recombinant protein after purification, wherein the purity of the recombinant protein is high and reaches more than 90%, the repeatability is good, and the recovery rate is high. The protein can be used as a candidate component of a vaccine for preventing neisseria meningitidis group B infection, has the advantages of soluble expression, easiness in purification, high purity, simple and convenient preparation method and the like, and has remarkable economic benefit.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a group B neisseria meningitidis recombinant bacterial hair protein Fim, and a preparation method and application thereof.
Background
Meningococcus, the scientific name neisseria meningitidis (n.meninyitidis), is the causative bacterium of epidemic cerebrospinal meningitis (epidemic encephalitis). It is a gram-negative diplococcus, which is present in the cerebrospinal fluid of patients in the acute or early stage of meningitis, mostly located in neutrophils, is kidney-shaped, and is arranged in double rows with concave surfaces facing each other. The virulent strains of meningococcus are seen to have microcapsules and pili under an electron microscope.
The incidence process of the neisseria meningitidis infection can be divided into 3 stages, namely ① pathogenic bacteria invade from the nasopharynx and adhere to the surface of nasopharyngeal mucosal epithelial cells by virtue of pili, ① pathogenic bacteria invade from the nasopharynx firstly and adsorb on the surface of the nasopharyngeal mucosal epithelial cells by virtue of the pili to cause local infection, ② bacteria invade blood flow to cause bacteremia accompanied by symptoms of chills, fever, vomiting, skin bleeding ecchymosis and the like, ③ bacteria invade the blood flow and multiply in quantity, and blood and lymph reach the cerebrospinal membrane to cause cerebrospinal membrane suppurative inflammation.
Meningococcus it has now been found that 13 serogroups, A, B, C, D, X, Y, Z, 29E, W135, L, H, I, K respectively, have not been able to be grouped in 1-5% of the worldwide epidemic. Most of the diseases causing human beings belong to A, B, C groups.
Currently, there are more studies on the prevention of group B meningococci, and two vaccines are on the market, the Trumenba vaccine approved in 10-29 months 2014 and the Bexsero vaccine approved in 1-23 months 2015 by FDA in the united states. The two vaccines are mainly used for individuals of 10-25 years old and can prevent invasive meningococcal disease caused by serogroup B. The Trumenba vaccine is a sterile suspension consisting of 2 variants of recombinant lipidated factor H binding protein (fHBP) from MenB. fHBP is one of a number of proteins located on the surface of meningococci and can help protect bacteria from attack by the host's immune defense system. fHBP can be divided into 2 immunologically distinct subfamilies, namely subfamily a and subfamily B. One of the Trumenba vaccines is from subfamily a of fHBP (a5) and the other is from subfamily B (B01). The susceptibility of MenB to complement-mediated antibody-dependent killing after Trumenba vaccination depends on the antigenic similarity of the bacteria and vaccine fHBP and the amount of fHBP expressed on the surface of the invading meningococcus.
The Bexsero vaccine contains 4 main components: factor H binding proteins, neisserial heparin binding antigens, neisserial adhesin a and NZ98/254 strain derived outer membrane vesicles.
Neisseria meningitidis invades the upper respiratory tract from the nasopharyngeal mucosa, further enters blood, and spreads with the blood to generate meningitis. The adhesion of neisseria meningitidis to the surface of upper respiratory cells is the first step in infection and if it can be blocked from adhering from the outset, it will not enter the blood and progress to meningitis. Currently, no relevant vaccine studies are on the market that block neisseria meningitidis fimbriae adhesion and thus prevent meningitis.
The pili on the surface of the B group meningococcus mediates the adhesion of bacteria on the surface of host susceptible cells, is beneficial to the colonization and propagation of the bacteria in a host body, and plays an important role in the infection, colonization and pathogenic processes of the bacteria. The meningococcus group B has multiple pili, and in the pili, the amino acid sequence of a part of pili is specific to the meningococcus group B, and the part of the sequence is highly conserved in the meningococcus group B and is greatly different from the meningococcus groups of other groups. The invention aims to screen a vaccine antigen for preventing group B meningococcus, and provides a basis for preparing a genetic engineering subunit vaccine by enlarging expression through a genetic engineering means.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing market lacks a vaccine for directly blocking the adhesion of the pili of neisseria meningitidis and further preventing meningitis.
The technical scheme for solving the technical problems comprises the following steps: provides a group B neisseria meningitidis recombinant Fim gene.
The invention provides a group B neisseria meningitidis recombinant pilin Fim gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.
Nucleotide sequence of recombinant pilin Fim gene of group 1B neisseria meningitidis of SEQ ID NO
atggcggaaggccagaaaagcgcggtgaccgaatattatctgaaccacggagaatggccgggcaacaacagcagcgcgggcgtggcgaccagcgcggatattaaaggcaaatatgtgaaaagcgtggaagtgaaaaacggcgtggtgaccgcgcagatggcgagcagcaacgtgaacaacgaaattaaaggcaaaaaactgagcctgtgggcgaaacgtcaggcgggcagcgtgaaatggttttgcggcctgccggtgacccgtgcggataacgcgaaagatgatgcggtgaccgcggcggcgaccggcaccgataaaattgataccaaacatctgccgagcacctgccgtgatgatagcagcgtggtgtgcattgaaaccccgccgaccgcgttttataaaaacaccctcgagcaccaccaccaccaccactga。
The invention also provides a group B neisseria meningitidis recombinant pilin Fim, the amino acid sequence of which is shown as SEQID NO. 2.
Amino acid sequence of recombinant pilin Fim of 2B group neisseria meningitidis
MAEGQKSAVTEYYLNHGEWPGNNSSAGVATSADIKGKYVKSVEVKNGVVTAQMASSNVNNEIKGKKLSLWAKRQAGSVKWFCGLPVTRADNAKDDAVTAAATGTDKIDTKHLPSTCRDDSSVVCIETPPTAFYKNT。
The invention also provides a preparation method of the group B neisseria meningitidis recombinant pilin Fim, which comprises the following steps:
a. plasmid construction
Connecting a gene with a nucleotide sequence of SEQ ID NO. 1 into an expression vector plasmid, constructing the plasmid and transferring the plasmid into host bacteria for induced expression;
b. ultrasonic bacteria breaking and centrifugation
B, collecting the thalli obtained in the step a, resuspending and uniformly mixing the thalli with a bacteria breaking solution, breaking the thalli by using ultrasonic after ice water bath, centrifuging at a high speed, and collecting a supernatant;
c. affinity purification with Ni column
Carrying out primary purification on the Ni affinity filler, using a liquid A balance chromatographic column, and using a liquid B for elution;
d. performing SP column affinity chromatography;
and D, purifying the target protein purified in the step C by using an SP column, using a C liquid equilibrium chromatographic column, and eluting by using a D liquid to obtain the recombinant pilin Fim.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the expression vector in the step a is pET28 a; the host bacterium is Escherichia coli.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the induction expression condition in the step a is 37 ℃ and 100-250 rpm; inducing isopropyl thiogalactoside with the concentration of 0.1-1 mM.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the bacterium breaking solution in the step B is 10-50 mM phosphate buffer solution with the pH value of 6-8 and 0.1-0.3M NaCl.
In the preparation method of the neisseria meningitidis group B recombinant bacterial hair protein Fim, the ultrasonic bacteria breaking in the step B is performed by using a Ningbo New technology ultrasonic equipment Co., Ltd, a JY 96-IIN crusher with the model number of 40%.
Further, in the preparation method of the neisseria meningitidis group B recombinant pilin protein Fim, ultrasonic is performed for 5 s-10 s during ultrasonic bacteria breaking in the step B, the ultrasonic bacteria breaking is stopped for 5 s-10 s, and the bacteria breaking is performed alternately for 10-60 min.
Further, in the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the bacterium breaking in the step B is performed for 2 times, and each time is 20 min.
Further, in the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the centrifugation speed in the step B is 10000-18000 g, and the centrifugation time is 30-60 min.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the Ni affinity filler in the step c is a nickel ion metal chelating affinity chromatography medium (Ni Sepharose High Performance, GE, the product number is 17-5268-02).
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution A in the step c comprises the following components: 10 to 50mM phosphate buffer solution having a pH of 6 to 8, and 0.1 to 0.3M NaCl.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution B in the step c is composed of 10-50 mM phosphate buffer solution with the pH value of 6-8, 0.1-0.3M NaCl and 0.5-1M imidazole.
In the preparation method of the Neisseria meningitidis group B recombinant pilin Fim, the SP column purification filler in the step d is SP Bestarose FF (Bogelong, Cat: AI 0011).
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution C in the step d is as follows: a 10-50 mM phosphate buffer solution having a pH of 6-8.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution D in the step D is 10-50 mM phosphate buffer solution with the pH value of 6-8 and 0.5-1M NaCl.
In the preparation method of the Neisseria meningitidis group B recombinant pilin Fim, the molecular weight of the recombinant protein obtained in the step d is 14-17 KD, and the isoelectric point pH is 7.4-9.4.
The invention also provides application of the group B neisseria meningitidis recombinant pilin Fim in preparation of a group B neisseria meningitidis vaccine.
Further, the application is the application for preparing the candidate antigen of the neisseria meningitidis vaccine group B.
The invention has the beneficial effects that:
the invention screens a section of recombinant protein gene in B group meningococcus pilus and names the recombinant pilin Fim gene, the invention discovers that the recombinant protein gene is a key molecule for adhesion and permanent planting of neisseria meningitidis for the first time, clones the recombinant protein to a protein expression vector for large-scale expression, and prepares the recombinant protein after purification, wherein the purity of the recombinant protein is high and reaches more than 90%, the repeatability is good, and the recovery rate is high. The recombinant pilin Fim and Freund adjuvant are injected into a BalB/C mouse together for immunization, and the result shows that the recombinant pilin Fim can effectively stimulate an organism to generate higher immune response, can effectively block the adhesion of Neisseria meningitidis aiming at the immune response, and has good sterilization effect proved by an antibody sterilization experiment.
Drawings
FIG. 1 shows the results of double digestion identification of pET28a/Fim/BL21(DE 3); lane 1-2 shows the results of the restriction enzyme assay, and the isolated fragments were about 5300bp and 435 bp; lane 3 shows a nucleic acid Mark (Thermo, SM 0311).
FIG. 2 shows the Fim protein induction identification result, lane 1 is Mark; lane 2 post-schizomycete pellet; lane 3 is post-schizomycete supernatant; lane 4 is whole broth; the result of the identification shows that the Fim protein is about 15.5kd and is identified as soluble expression.
FIG. 3 shows the result of Ni column affinity chromatography purification.
FIG. 4 shows the SDS-PAGE result after Ni column affinity chromatography, and lane 1 is the upper column sample; lane 2 is flow through; lanes 3-5, 50mM imidazole eluting heteroproteins; lane 6 is Mark; lanes 7-8 are 300mM imidazole-eluted Fim proteins.
FIG. 5 shows the results of SP column chromatography purification.
FIG. 6 shows SDS-PAGE electrophoresis results after SP column chromatography, wherein lane 1 is an upper Ni column sample; lane 2 is Ni column flow through; lane 3 is 300mM imidazole-eluted Fim protein; lane 4 is Mark; lane 5 is an upper SP column sample; lane 6 SP column flow through; lane 7 shows the 90% pure Fim protein eluted.
Detailed Description
The invention provides a group B neisseria meningitidis recombinant pilin Fim gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.
The meningococcus group B has multiple pili, and in the pili, the amino acid sequence of a part of pili is specific to the meningococcus group B, and the part of the sequence is highly conserved in the meningococcus group B and is greatly different from the meningococcus groups of other groups. The invention particularly screens out a gene segment shown as SEQ ID NO. 1, named as Fim gene, finds that the gene segment is a key molecule for adhesion and permanent planting of neisseria meningitidis, clones the gene segment to a protein expression vector for large-scale expression, prepares recombinant protein after purification, and can be used as a candidate antigen in preparation of neisseria meningitidis vaccine group B.
The invention also provides a group B neisseria meningitidis recombinant pilin Fim, the amino acid sequence of which is shown as SEQID NO. 2.
Further, the invention also provides a preparation method of the group B neisseria meningitidis recombinant pilin Fim, which comprises the following steps:
a. plasmid construction
Connecting a gene with a nucleotide sequence of SEQ ID NO. 1 into an expression vector plasmid, constructing the plasmid and transferring the plasmid into host bacteria for induced expression;
b. ultrasonic bacteria breaking and centrifugation
B, collecting the thalli obtained in the step a, resuspending and uniformly mixing the thalli with a bacteria breaking solution, breaking the thalli by using ultrasonic after ice water bath, centrifuging at a high speed, and collecting a supernatant;
c. affinity purification with Ni column
Carrying out primary purification on the Ni affinity filler, using a liquid A balance chromatographic column, and using a liquid B for elution;
d. performing SP column affinity chromatography;
and D, purifying the target protein purified in the step C by using an SP column, using a C liquid equilibrium chromatographic column, and eluting by using a D liquid to obtain the recombinant pilin Fim.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the expression vector in the step a is pET28 a; the host bacterium is Escherichia coli.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the induction expression condition in the step a is 37 ℃ and 100-250 rpm; inducing isopropyl thiogalactoside with the concentration of 0.1-1 mM.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the bacterium breaking solution in the step B is 10-50 mM phosphate buffer solution with the pH value of 6-8 and 0.1-0.3M NaCl.
In the preparation method of the neisseria meningitidis recombinant pilin Fim, in order to more completely lyse bacteria and ensure that a sample is not damaged, the ultrasonic bacteria breaking in the step B is performed by using a crusher of Ningbo New technology ultrasonic equipment Limited company with the model number of JY 96-IIN, and the power is 40%.
Further, in the preparation method of the neisseria meningitidis group B recombinant pilin protein Fim, ultrasonic is performed for 5 s-10 s during ultrasonic bacteria breaking in the step B, the ultrasonic bacteria breaking is stopped for 5 s-10 s, and the bacteria breaking is performed alternately for 10-60 min.
Furthermore, in order to sufficiently cool the sample and prevent the influence caused by overhigh temperature, in the preparation method of the recombinant pilin protein Fim of neisseria meningitidis group B, the bacterium breaking in the step B is performed for 20min for 2 times.
Further, in the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the centrifugation speed in the step B is 10000-18000 g, and the centrifugation time is 30-60 min.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the Ni affinity filler in the step c is a nickel ion metal chelating affinity chromatography medium (Ni Sepharose High Performance, GE, the product number is 17-5268-02).
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution A in the step c comprises the following components: 10 to 50mM phosphate buffer solution having a pH of 6 to 8, and 0.1 to 0.3M NaCl.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution B in the step c is composed of 10-50 mM phosphate buffer solution with the pH value of 6-8, 0.1-0.3M NaCl and 0.5-1M imidazole.
In the preparation method of the recombinant pilin protein Fim of the neisseria meningitidis group B, the SP column purification filler in the step d is SPBestarose FF (Bogelong, Cat: AI0011) in order to maintain the charge property and state of the target protein, so that the purification is easy and the target protein is better obtained.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution C in the step d is as follows: a 10-50 mM phosphate buffer solution having a pH of 6-8.
In the preparation method of the neisseria meningitidis group B recombinant pilin Fim, the solution D in the step D is 10-50 mM phosphate buffer solution with the pH value of 6-8 and 0.5-1M NaCl.
In the preparation method of the Neisseria meningitidis group B recombinant pilin Fim, the molecular weight of the recombinant protein obtained in the step d is 14-17 KD, and the isoelectric point pH is 7.4-9.4.
The invention also provides application of the group B neisseria meningitidis recombinant pilin Fim in preparation of a group B neisseria meningitidis vaccine.
Further, the application is the application for preparing the candidate antigen of the neisseria meningitidis vaccine group B.
The following examples are given to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the present invention to the examples.
Examples Neisseria meningitidis group B as used herein was purchased from ATCC (Albrecht and Ghon) Murray (ATCC) in usa
13090) ); plasmid pET-28a was purchased from GE Healthcare Life sciences; coli strain BL21(DE3) was purchased from Shanghai super research Biotech, Inc.; DNA Marker, restriction enzyme Nco I and Xho I, T4 DNA Ligase, protein Marker is Thermo Fisher product; the plasmid extraction kit, the gel recovery kit, the bacterial genome extraction kit and the ultrathin recovery kit are products of Tiangen Biochemical technology (Beijing) Co., Ltd; the rest of the reagents are common commercial products.
Example 1 construction and characterization of recombinant plasmid pET28a/Fim of Fim Gene
The specific operation steps are as follows:
(1) according to the whole genome sequence of the neisseria meningitidis group B, a candidate antigen Fim (SEQ ID NO:2) with protective immune response is screened out by applying reverse vaccinology.
(2) According to the amino acid sequence of Fim, optimization of escherichia coli preference codon is carried out to obtain a target gene fragment, the sequence is SEQ ID NO. 3 (base sequence of restriction enzyme cutting site is underlined).
Nucleotide sequence of SEQID NO. 3 target gene
ccatggcggaaggccagaaaagcgcggtgaccgaatattatctgaaccacggagaatggccgggcaacaacagcagcgcgggcgtggcgaccagcgcggatattaaaggcaaatatgtgaaaagcgtggaagtgaaaaacggcgtggtgaccgcgcagatggcgagcagcaacgtgaacaacgaaattaaaggcaaaaaactgagcctgtgggcgaaacgtcaggcgggcagcgtgaaatggttttgcggcctgccggtgacccgtgcggataacgcgaaagatgatgcggtgaccgcggcggcgaccggcaccgataaaattgataccaaacatctgccgagcacctgccgtgatgatagcagcgtggtgtgcattgaaaccccgccgaccgcgttttataaaaacaccctcgagcaccaccaccaccaccacctcgagcaccaccaccaccaccac。
(3) The target gene is synthesized, and is inserted into an expression plasmid pET28a (completed by Wuhan Kingkunrei bioengineering, Co., Ltd.) through Nco I and Xho I enzyme cutting sites, the sequencing result of the plasmid is compared with the sequence information submitted for synthesis, and the nucleotide sequence is completely the same.
(4) After dissolving the synthetic plasmid, transferring into competent cells of Escherichia coli BL21(DE3), carrying out ice bath for 30min, carrying out heat shock for 90s at 42 ℃, and carrying out ice bath rapidly for 3 min. Adding 1ml SOC culture medium, mixing, and shaking in 37 deg.C shaker at 220rpm for 45 min.
(5) 100 mul of bacterial liquid was spread on a kanamycin-resistant LB plate and cultured in a 37 ℃ incubator for 16 hours.
(6) And (3) screening and identifying a pET28a/Fim/BL21(DE3) positive recombinant plasmid. The specific operation is as follows:
a. selecting a single colony well separated on a transformation plate, inoculating the single colony in a kanamycin + resistant LB culture medium, and carrying out shaking culture at 37 ℃ for overnight;
b. plasmid extraction: reference is made to the plasmid extraction kit instructions;
c. carrying out double enzyme digestion on plasmid DNA by Nco I and Xho I; the double digestion reaction system is shown in table 1: enzyme digestion is carried out for 1h at 37 ℃;
TABLE 1 double digestion reaction System
d. The double restriction enzyme cutting result is detected by 1.5 percent agarose gel electrophoresis, and the result is shown in figure 1, which indicates that the recombinant plasmid is successfully transformed.
Example 2 the specific operation steps of induction expression, purification and identification of expression form of recombinant Fim in prokaryotic expression system-escherichia coli are as follows:
(1) adding 100 μ l of overnight cultured pET28a/Fim/BL21(DE3) bacterial liquid into 10mL of kanamycin + resistant LB culture medium, culturing overnight at 220rpm 37 ℃, adding 200 μ l of overnight cultured bacterial liquid into 20mL of kanamycin + resistant LB culture medium, culturing at 220rpm 37 ℃ for 2h, activating twice to OD600 of 0.8, adding IPTGIsopropyl- β -D-thioxo moiety LactosideMu.l of the recombinant plasmid was added to a final concentration of 0.5mM, and the mixture was then allowed to stand on a shaker at 220rpm and 37 ℃ for induced expression for 4 hours.
(2) Taking out the bacteria liquid after induction expression, centrifuging for 15min at 5000g, discarding the supernatant, adding 3ml of bacteria-breaking liquid (50mM PB, 0.3M NaCl, pH7.4), mixing well, performing ultrasonic lysis in ice bath for 10min, centrifuging for 30min at 12000g at 4 ℃, and separating the supernatant and the precipitate.
(3) Processing samples
Adding 3ml of bacteria breaking liquid into the sediment for resuspension, respectively taking 40 mul of bacteria breaking liquid, supernatant and resuspended sediment, adding 10 mul of 5X protein loading buffer (Bio-engineering, product number: C508320-0010), centrifuging at 100 ℃ for 10min and 12000g for 3 min.
(4) SDS-PAGE electrophoresis
And respectively taking 10 mu l of the treated lysate liquid, supernatant and precipitate, carrying out 15% SDS-PAGE electrophoresis, carrying out Coomassie brilliant blue staining, and then scanning by a gel scanning imaging system (FUSION-FX6.EDGE V.070). The results showed that pET28a/Fim/BL21(DE3) was soluble expression (FIG. 2).
Example 3 preparation of Fim antigen
The specific operation steps are as follows:
(1) amplifying culture to obtain protein
30mL of overnight-cultured pET28a/Fim/BL21(DE3) bacterial solution was added to 3L of kanamycin + resistant TB medium (containing KH/L: KH)2PO42.31g、K2HPO412.54g, tryptone 12g, yeast extract 24g, glycerol 4ml), cultured at 37 ℃ at 220rpm 2, and added 1M when the culture reached an OD600 of 0.8Isopropyl- β -D-thiogalactoside1.5ml, to a final concentration of 0.5mM, was induced at 220rpm for 4h at 37 ℃. The induced bacterial liquid is centrifuged for 15min at 8000g to collect thalli, 160ml of bacteria-breaking liquid (same as example 2) is added to resuspend the thalli, the bacterial liquid is subjected to ultrasonic lysis for 3 times (20min), and centrifuged for 30min at 12000g to collect supernatant.
(2) Purification by Fim
a. Affinity chromatography on Ni column
Taking supernatant of the bacteria-breaking liquid, and filtering the supernatant through a 0.45 mu m filter membrane for later use. The Ni column affinity chromatography column was equilibrated with solution A (50mM PB, 0.3M NaCl, pH7.4), the filtered supernatant was applied, and then the impurities were eluted with 20% solution B (50mM PB, 0.3M NaCl, 0.5M imidazole, pH7.4) + 80% solution A, and the target protein was eluted with 60% solution B + 40% solution A. The chromatogram is shown in FIG. 3, and the electrophoresis results are shown in FIG. 4.
b. SP column chromatography
The protein eluted in (1) was added to 2.5 times the volume of ultrapure water, and then added to 11.5 times the volume of liquid C (20mM PB, pH7.4) to dilute it for further use. And (3) balancing the SP chromatographic column by using a solution C, sampling and loading a sample, washing and balancing the solution C, eluting by using a solution D (20mM PB, 0.5M NaCl, pH7.4), collecting eluted target protein, and storing at 4 ℃ for later use. The chromatogram is shown in figure 5, the protein peak of 2400mAu is eluted, the identification electrophoresis result is shown in figure 6, the target protein with the purity of more than 90% is obtained, and the sequence is shown in SEQ ID NO: 4, the requirements of subsequent experiments are met.
SEQ ID NO: 4 amino acid sequence of recombinant Fim
MAEGQKSAVTEYYLNHGEWPGNNSSAGVATSADIKGKYVKSVEVKNGVVTAQMASSNVNNEIKGKKLSLWAKRQAGSVKWFCGLPVTRADNAKDDAVTAAATGTDKIDTKHLPSTCRDDSSVVCIETPPTAFYKNTLEHHHHHH。
EXAMPLE 4 immunization of animals
Balb/C mice, female, 8-10 weeks old, were purchased from Beijing Huafukang Biotech GmbH. The groups were divided into an immunization group (protein Fim antigen + adjuvant), a negative control group (adjuvant) and a blank control group, with 30 individuals each.
(1) First immunization, 50 μ g antigen and freund's complete adjuvant 1:1 mix (total volume 1mL), axilla (50. mu.l. times.2), groin (50. mu.l. times.2), and neck and back subcutaneous injection (100. mu.l).
(2) Second immunization, day 14 with a second immunization, 50 μ g antigen and Freund's incomplete adjuvant 1:1 mixing (total volume 1mL), and injecting the mixture in the same way as the immunization route;
(3) third immunization, carried out on day 21, with 50 μ g antigen mixed with PBS (total volume 1mL), injected in the same amount as the route of immunization;
(4) the fourth immunization, carried out on day 28, was carried out in the same amount as the route of immunization.
EXAMPLE 5 detection of antibodies
5 days after the fourth immunization, blood was collected from Balb/C mice, serum was isolated, and changes in Fim-specific IgG levels were detected by Elisa.
The Elisa detection reagent comprises:
(1) coating liquid: na (Na)2CO31.6g，NaHCO32.9g in 1L ddH2O, adjusting the pH value to 9.6;
(2)PBS：NaCl 8g，KCl 0.2g，Na2HPO4 1.42g，K2PO40.27g in 1L ddH2O；
(3) Washing liquid: dissolve 500. mu.l Tween 20 in 1L PBS;
(4) sealing liquid: 1g BSA in 100mL PBS;
(5) antibody dilution: 0.05g Tween 20, 0.5g BSA, dissolved in 100mL PBS;
(6) color development liquid:
a: solid TMB was dissolved in DMSO to a final concentration of 1mg/mL and stored at 4 ℃ in the dark.
B substrate buffer solution: na (Na)2HPO41.42g was dissolved in 100mL ddH2O, adjusting pH to 5.0 using citric acid;
c: storing with 30% hydrogen peroxide at 4 deg.C in dark place;
the color developing solution A, the color developing solution B and the color developing solution C are prepared according to the ratio of 100:900:1, and the color developing solution is ready to use.
Stopping liquid: 10.87mL of concentrated sulfuric acid was added to 60mL of ddH2And cooling the solution in the O solution, and then, metering the volume to 100 mL.
The operation steps are as follows:
elisa detects the antibody titer generated by mice immunized with Fim recombinant protein:
(1) diluting the Fim protein to 2 mu g/mL by using a coating solution;
(2) coating: adding the diluted Fim protein solution into an enzyme label plate, incubating for 2h at 37 ℃ in a hole of 100 mu l, and washing for 4 times by PBST;
(3) and (3) sealing: adding 300 mul/hole of blocking liquid into an enzyme label plate, incubating for 2h at 37 ℃, washing for 4 times by PBST, and storing for later use at 4 ℃;
(4) serial double dilutions of serum from 1:32768 to 1: 1048576;
(5) taking the coated ELISA plate, sequentially adding diluted serum and 100 mul/hole, making multiple holes, incubating at 37 ℃ for 1h, and washing by PBST for 4 times;
(6) adding HRP-labeled goat anti-mouse IgG (manufactured and assigned with a product number of D110087-0100) diluted by antibody diluent of 1:10000 into the mixture, incubating the mixture at 37 ℃ for 30min, and washing the mixture with PBST for 4 times;
(7) adding 100 mul/hole of substrate color development solution, and incubating at 37 deg.C for 10 min;
(8) adding 50 mul/hole of stop solution, immediately placing on an enzyme-linked immunosorbent assay (ELISA) instrument, and measuring an OD value at a wavelength of 450 nm;
(9) and (5) judging a result: the A sample/A negative is more than or equal to 2.1 and is positive.
As a result: detecting that the titer of the antibody generated by the Fim protein antigen immunized mouse reaches 1: 262144, a binder resin; the antibody positive rate after immunization reaches 100%, which indicates that the Fim recombinant protein can enable the immunized mice to generate antibodies, and the antibody titer is shown in table 2.
TABLE 2 antibody Titers test
Example 6 evaluation of serum antibody bactericidal Activity after immunization with Fim recombinant protein
The specific operation steps are as follows:
(1) neisseria meningitidis was inoculated into chocolate plates at 37 ℃ with 5% CO2The incubator was inverted for 16 hours. After culturing, 1 single colony was selected and streaked on a new plate at 37 ℃ with 5% CO2Culturing for 18 h;
(2) scraping lawn, and suspending the lawn in PBS to make OD value at 600nm be 0.4;
(3) the complement of the serum to be detected is inactivated at 56 ℃ for 40min, and the serum is diluted by PBS in multiple ratio to obtain diluted serum with the dilution degrees of 2, 4, 8, 16, 32, 64, 128, 256 and 512.
(4) Mixing 25. mu.l of diluted serum with 12.5. mu.l of complement serum (guinea pig) and 12.5. mu.l of bacterial suspension; three controls were also set, including complement-free serum, antibody-free serum and inactivated complement serum (40 min at 56 ℃). (all groups are provided with 10 compound holes respectively)
(5) The mixed bacterial liquid was cultured at 220rpm and 37 ℃ for 60 min. 50 μ l of TTC agar medium (Solarbio, LA1220) melted and cooled to about 45 ℃ in each well, 5% CO at 37 ℃2The results were observed after 24h of incubation.
As a result: when the approximate number of colonies in the wells was close to about half or more of the negative control wells with reference to the negative control (containing no complement serum), the wells were judged as non-bactericidal, i.e., only when the approximate number of colonies in the wells was significantly less than the negative control wells, the wells were judged as bactericidal positive (as shown in table 3).
TABLE 3 serum antibody Sterilization test results
The embodiment shows that the Fim protein is a key molecule for adhesion and permanent planting of the neisseria meningitidis for the first time, the Fim protein is cloned to a protein expression vector for large-scale expression, and the protein is purified to prepare the recombinant protein, wherein the purity of the recombinant protein is high and reaches more than 90%, the repeatability is good, and the recovery rate is high. The immune response of the recombinant pilin can effectively block the adhesion of neisseria meningitidis, and the recombinant pilin Fim can be used as a candidate antigen in the preparation of group B neisseria meningitidis vaccines, and has a good application prospect.
Sequence listing
<110> Sichuan university Hospital in western China
<120> group B neisseria meningitidis recombinant bacterial hair protein Fim and preparation method and application thereof
<130> A200119K (preface)
<141>2020-03-17
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>435
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atggcggaag gccagaaaag cgcggtgacc gaatattatc tgaaccacgg agaatggccg 60
ggcaacaaca gcagcgcggg cgtggcgacc agcgcggata ttaaaggcaa atatgtgaaa 120
agcgtggaag tgaaaaacgg cgtggtgacc gcgcagatgg cgagcagcaa cgtgaacaac 180
gaaattaaag gcaaaaaact gagcctgtgg gcgaaacgtc aggcgggcag cgtgaaatgg 240
ttttgcggcc tgccggtgac ccgtgcggat aacgcgaaag atgatgcggt gaccgcggcg 300
gcgaccggca ccgataaaat tgataccaaa catctgccga gcacctgccg tgatgatagc 360
agcgtggtgt gcattgaaac cccgccgacc gcgttttata aaaacaccct cgagcaccac 420
caccaccacc actga 435
<210>2
<211>136
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Met Ala Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His
1 5 10 15
Gly Glu Trp Pro Gly Asn Asn Ser Ser Ala Gly Val Ala Thr Ser Ala
20 25 30
Asp Ile Lys Gly Lys Tyr Val Lys Ser Val Glu Val Lys Asn Gly Val
35 40 45
Val Thr Ala Gln Met Ala Ser Ser Asn Val Asn Asn Glu Ile Lys Gly
50 55 60
Lys Lys Leu Ser Leu Trp Ala Lys Arg Gln Ala Gly Ser Val Lys Trp
65 70 75 80
Phe Cys Gly Leu Pro Val Thr Arg Ala Asp Asn Ala Lys Asp Asp Ala
85 90 95
Val Thr Ala Ala Ala Thr Gly Thr Asp Lys Ile Asp Thr Lys His Leu
100 105 110
Pro Ser Thr Cys Arg Asp Asp Ser Ser Val Val Cys Ile Glu Thr Pro
115 120 125
Pro Thr Ala Phe Tyr Lys Asn Thr
130 135
<210>3
<211>458
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ccatggcgga aggccagaaa agcgcggtga ccgaatatta tctgaaccac ggagaatggc 60
cgggcaacaa cagcagcgcg ggcgtggcga ccagcgcgga tattaaaggc aaatatgtga 120
aaagcgtgga agtgaaaaac ggcgtggtga ccgcgcagat ggcgagcagc aacgtgaaca 180
acgaaattaa aggcaaaaaa ctgagcctgt gggcgaaacg tcaggcgggc agcgtgaaat 240
ggttttgcgg cctgccggtg acccgtgcgg ataacgcgaa agatgatgcg gtgaccgcgg 300
cggcgaccgg caccgataaa attgatacca aacatctgcc gagcacctgc cgtgatgata 360
gcagcgtggt gtgcattgaa accccgccga ccgcgtttta taaaaacacc ctcgagcacc 420
accaccacca ccacctcgag caccaccacc accaccac 458
<210>4
<211>144
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Met Ala Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His
1 5 10 15
Gly Glu Trp Pro Gly Asn Asn Ser Ser Ala Gly Val Ala Thr Ser Ala
20 25 30
Asp Ile Lys Gly Lys Tyr Val Lys Ser Val Glu Val Lys Asn Gly Val
35 40 45
Val Thr Ala Gln Met Ala Ser Ser Asn Val Asn Asn Glu Ile Lys Gly
50 55 60
Lys Lys Leu Ser Leu Trp Ala Lys Arg Gln Ala Gly Ser Val Lys Trp
65 70 75 80
Phe Cys Gly Leu Pro Val Thr Arg Ala Asp Asn Ala Lys Asp Asp Ala
85 90 95
Val Thr Ala Ala Ala Thr Gly Thr Asp Lys Ile Asp Thr Lys His Leu
100 105 110
Pro Ser Thr Cys Arg Asp Asp Ser Ser Val Val Cys Ile Glu Thr Pro
115 120 125
Pro Thr Ala Phe Tyr Lys Asn Thr Leu Glu His His His His His His
130 135 140
Claims (10)
- Group B neisseria meningitidis recombinant Fim gene, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 1.
- 2. The gene-expressed neisseria meningitidis group B recombinant pilin Fim of claim 1, wherein: the amino acid sequence is shown in SEQ ID NO. 2.
- 3. The method of producing the neisseria meningitidis group B recombinant pilin Fim of claim 2, comprising the steps of:a. plasmid constructionConnecting a gene with a nucleotide sequence of SEQ ID NO. 1 into an expression vector plasmid, constructing the plasmid and transferring the plasmid into host bacteria for induced expression;b. ultrasonic bacteria breaking and centrifugationB, collecting the thalli obtained in the step a, resuspending and uniformly mixing the thalli with a bacteria breaking solution, breaking the thalli by using ultrasonic after ice water bath, centrifuging at a high speed, and collecting a supernatant;c. affinity purification with Ni columnCarrying out primary purification on the Ni affinity filler, using a liquid A balance chromatographic column, and using a liquid B for elution;d. performing SP column affinity chromatography;and D, purifying the target protein purified in the step C by using an SP column, using a C liquid equilibrium chromatographic column, and eluting by using a D liquid to obtain the recombinant pilin Fim.
- 4. The method for preparing the Neisseria meningitidis group B recombinant pilin Fim according to claim 3, characterized in that: the expression vector in the step a is pET28 a; the host bacterium is Escherichia coli.
- 5. The method for preparing the Neisseria meningitidis group B recombinant pilin Fim according to claim 3, characterized in that: the condition of induced expression in the step a is 37 ℃, and 100-250 rpm; inducing isopropyl thiogalactoside with the concentration of 0.1-1 mM.
- 6. The method for preparing the Neisseria meningitidis group B recombinant pilin Fim according to claim 3, characterized in that: the bacterium breaking solution in the step b is 10-50 mM phosphate buffer solution with the pH value of 6-8 and 0.1-0.3M NaCl; and when the ultrasonic bacteria is broken for 5 s-10 s, stopping the ultrasonic treatment for 5 s-10 s, and alternately breaking the bacteria for 10-60 min.
- 7. The method for preparing the Neisseria meningitidis group B recombinant pilin Fim according to claim 3, characterized in that: and c, the centrifugation speed of the step b is 10000-18000 g, and the centrifugation time is 30-60 min.
- 8. The method for preparing the Neisseria meningitidis group B recombinant pilin Fim according to claim 3, characterized in that: the composition of the solution A in the step c is as follows: 10-50 mM phosphate buffer solution with pH of 6-8, 0.1-0.3M NaCl; the solution B comprises 10-50 mM phosphate buffer solution with pH of 6-8, 0.1-0.3M NaCl, and 0.5-1M imidazole.
- 9. The method for preparing the Neisseria meningitidis group B recombinant pilin Fim according to claim 3, characterized in that: the solution C in the step d is: 10-50 mM phosphate buffer solution with pH of 6-8; the solution D is 10-50 mM phosphate buffer solution with pH of 6-8 and 0.5-1M NaCl.
- 10. Use of the neisseria meningitidis group B recombinant pilin Fim of claim 2 in the manufacture of a neisseria meningitidis group B vaccine.
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| CN114350696A (en) * | 2021-12-21 | 2022-04-15 | 四川大学华西医院 | Recombinant vector of soluble helicobacter pylori vaccine recombinant antigen UreA, expression and purification method and application thereof |
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