CN118085108A - Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and preparation method and application thereof - Google Patents
Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, and the amino acid sequence of the antigen protein is shown as SEQ ID NO. 1. Wherein mHla is a nontoxic mutant of staphylococcus aureus alpha hemolysin, which can be used as carrier protein of epitope to form heptamer; epiVac contains three immunodominant epitopes of klebsiella pneumoniae, and is formed by connecting the immunodominant epitopes through a linker. The invention also discloses a preparation method and application of the antigen protein. The antigen protein prepared by the method can effectively stimulate organisms to generate high-efficiency humoral response and cellular immune response, can provide obvious protection effect on killing dose of Klebsiella pneumoniae infection, and can be used as candidate antigens of Klebsiella pneumoniae vaccine.
Description
Technical Field
The invention belongs to the field of biotechnology pharmacy, and relates to a klebsiella pneumoniae vaccine candidate antigen and a preparation method thereof, and application of the antigen protein in preparation of klebsiella pneumoniae recombinant protein vaccine.
Background
In 2017, the world health organization first released a list of 12 key pathogens that severely endangered human health, with enterobacteriaceae bacteria represented by klebsiella pneumoniae (Klebsiella pneumoniae, KP) being listed as a paramount class (Tacconelli E, et al, lancet select dis 2018). KP is a gram-negative bacterium and is one of the most common opportunistic pathogens in clinic. KP can be planted in the intestinal tract, nasopharynx, armpit and other parts, wherein the digestive tract is the most main planting part. The colonic colonisation rate in western countries is 5% -35% and in asia countries are generally higher, and individual countries (e.g. malaysia) are even up to more than 85% (Zhang Xin, et al, tuberculosis and respiratory journal 2020). KP can cause infections at various parts of the whole body, and is common in elderly patients, malnutrition, chronic diseases and patients with systemic failure, and can cause systemic or local infections such as pneumonia, urinary tract infection, meningitis, septicemia, etc. (Lee CR, et al front CELL INFECT microbiol 2017). According to different virulence and pathogenic characteristics, KP is divided into two types at present, one type is classical Klebsiella pneumoniae (cKP), which mainly causes hospital acquired infection, such as pneumonia, urinary tract infection, septicemia and the like, has high drug resistance rate, and is common to people with basic diseases or low immunity. The other group is klebsiella pneumoniae (hvKP) which is a high virulence klebsiella pneumoniae and mainly causes infection of healthy people without basic diseases in communities, most commonly liver abscess, 66% of which are caused by KP infection (Russo TA, et al Clin Microbiol Rev.2019).
In recent years, the drug resistance situation of KP against various common antibiotics is more and more severe, and the drug resistance monitoring result of CHINET Chinese bacteria in 2022 shows that the separation rate of KP is the second place of gram negative bacteria, which is inferior to that of Escherichia coli, and reaches 13.99% in 2022. The drug resistance rate of the anti-aging agent to ampicillin is up to 88.5.8%, and the drug resistance rate of the anti-aging agent to piperacillin is over 50%. Particularly, with the wide clinical application of carbapenem medicines, the detection rate of the carbapenem-resistant klebsiella pneumoniae (CRKP) is increased year by year, the drug resistance rate of KP to imipenem and meropenem in China is respectively increased from about 3% in 2005 to 22.6% and 24.2% in 2022, and the modes are very severe (https:// www.chinets.com/Document). Furthermore, KP infection has extremely high mortality rates, statistically: patients with community pneumonia caused by KP of 22-32% need ICU treatment, and the death rate is up to 45% -72%; in addition, KP accounts for 5-20% of cases of sepsis due to gram-negative bacterial infection, with mortality rates as high as 27.4% -37% (Paczosa MK, et al, microbiol Mol Biol Rev.2016). More importantly, research shows that the KP infection mortality rate sensitive to carbapenem antibacterial drugs is 20% -30%, and CRKP infection mortality rate is remarkably increased by 40% -70% (Iredell J, et al BMJ.2016), and CRKP is called "super bacterial king" because of super drug resistance and pathogenicity. Considering that the KP drug resistance situation is very severe, and especially the wide popularity of CRKP makes antibiotic treatment difficult, the development of new effective control means is urgent, and vaccine development is one of the most promising strategies.
Since the advent of KP vaccine-related studies in the 70 s of the 20 th century, many different types of vaccine studies have emerged, early studies being based on inactivated, attenuated and bacterin-split vaccines, which are complex in composition, difficult to control quality, and may have residual toxicity, failing to enter clinical studies based on safety considerations (Ahmad TA, et al vaccine.2012). Later, research into ribosome vaccines was started, but since it is an intracellular component, immunoprotection was limited, and intensive studies have not been performed. And secondly, polysaccharide vaccine, wherein antigens adopted by the polysaccharide vaccine mainly comprise capsular polysaccharide and LPS, but as the capsular polysaccharide (K-antigen) of klebsiella pneumoniae has more than 80 serotypes, the LPS (O-antigen) has 12 serotypes, the K-antigen and the O-antigen have great variation among different serotypes, and although researches show that the vaccine has good immunogenicity and safety in human body experiments, and the passive immunity also has better protection effect, the cross protection capability on other serotypes is limited, and the application of the vaccine is objectively limited (Jenney AW, et al J Clin Microbiol.2006). Recent studies have focused mainly on recombinant protein vaccine studies, where various secreted proteins and outer membrane proteins exhibit a protective effect against KP infection, such as outer membrane proteins (OmpA, ompK36, fepA, ompK17, ompW), colicin I receptor proteins, adhesin MrkD proteins, pilin, cell surface iron regulatory proteins, toxoids, etc. (Zhang BZ, et al front immunol 2021), are currently the most promising types of vaccines.
Alpha hemolysin (Hla) is a member of the perforin family secreted by staphylococcus aureus, hla can self-assemble into a heptameric structure on the host cell membrane, leading to cell lysis and death, and a Hla mutant (mHla) lacking two β fragments forming a pore-like structure retains the ability to assemble into a heptamer, but loses its biological activity (Zou JT, et al front immunol 2021). mHla can be used as a universal carrier protein to improve the immunogenicity of protein antigens. First, the formation of oligomers increases the size of the antigenic protein. Second, fusion with mHla facilitates exposure of hidden epitopes on antigen proteins (Zou JT, et al plos pathway.2021).
Disclosure of Invention
Aiming at the high infection rate, high pathogenicity and high drug resistance of the klebsiella pneumoniae, the invention provides a klebsiella pneumoniae vaccine fusion protein antigen mHla-EpiVac which can be applied to the preparation of a klebsiella pneumoniae recombinant subunit vaccine.
The klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac provided by the invention has an amino acid sequence shown in SEQ ID NO. 1. Preferably, the fusion antigen contains three immunodominant epitopes of klebsiella pneumoniae origin.
The amino acid sequences of the three immunodominant epitopes are FepA to 423, shown as SEQ ID NO. 2, ompA148 to 165, shown as SEQ ID NO. 3, and OmpW144 to 163, shown as SEQ ID NO. 4.
The invention also provides a preparation method of the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, which mainly comprises the following steps:
1) mHla-EpiVac synthesis and subcloning;
2) mHla-EpiVac;
3) mHla-EpiVac protein antigen is prepared;
4) mHla-EpiVac protein purification.
Specifically, step 1) comprises: mHla was ligated to FepA396-423 sequence shown in SEQ ID NO. 2 with a flexible strand, then ligated to OmpW144-163 sequence shown in SEQ ID NO. 4 with a flexible strand, and then ligated to OmpA148-165 sequence shown in SEQ ID NO. 3 with a flexible strand to form fusion antigen mHla-EpiVac.
Step 1) further comprises the construction of a recombinant expression plasmid using the ampicillin-resistant prokaryotic expression plasmid pGEX-6 p-1.
Step 3) comprises culturing the induced protein obtained in step 2) in an amplified manner.
Step 4) comprises: collecting genetically engineered bacteria expressing mHla-EpiVac; breaking bacteria according to high pressure and centrifuging; cation exchange; sequential combinations of hydrophobic chromatography purify mHla-EpiVac.
The klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac can be used for preparing an immune preparation for preventing or treating klebsiella pneumoniae.
The invention also provides a klebsiella pneumoniae injection immune preparation, which contains the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and any one or any combination of the following adjuvants: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete freund's adjuvant, incomplete freund's adjuvant, mycobacterial bcg adjuvant.
As above, the present invention links mHla and FepA396-423 using flexible LINKERGGGGS strands, then links OmpW144-163 using flexible LINKER GGGGS, and finally links OmpA148-165 using flexible LINKER GSGGSG by fusion expression of 3 epitopes and mHla. The fusion protein is named mHla-EpiVac, and the amino acid sequence of the fusion protein is SEQ ID NO. 1.
The invention preferably adopts a prokaryotic expression plasmid pGEX-6p-1 to construct a recombinant expression plasmid, and the vector has ampicillin resistance and can be used for screening positive recombinants. To facilitate subsequent industrialization, no additional tag sequences were introduced at both ends of the fusion protein.
The invention provides a purification method of klebsiella pneumoniae recombinant protein antigen mHla-EpiVac. The main technical scheme is as follows: collecting genetically engineered bacteria expressing mHla-EpiVac; breaking bacteria according to high pressure and centrifuging; cation exchange; sequential combinations of hydrophobic chromatography purify mHla-EpiVac. The method has the advantages of simple process, high purity of the obtained target protein, easy amplification, good repeatability and good recovery rate.
The recombinant protein is cloned and expressed by adopting a genetic engineering technology, is convenient for separation and purification, can be directly matched with an adjuvant (such as an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, an MF59, a complete Freund adjuvant, an incomplete Freund adjuvant, a mycobacteria BCG vaccine adjuvant and the like) for use, and is suitable for injection immunization.
The genetic engineering recombinant mHla-EpiVac protein has the following advantages:
1) The recombinant mHla-EpiVac antigen protein contains three immunodominant epitopes of three antigens of klebsiella pneumoniae, and can simultaneously generate specific immune responses aiming at the three proteins;
2) The recombinant mHla-EpiVac antigen protein can be expressed in a prokaryotic expression system, namely escherichia coli, and has low cost and high yield;
3) When pGEX-6p-1 vector is selected, mHla-EpiVac recombinant protein is expressed in soluble form;
4) mHla-EpiVac has mild purification conditions, simple steps, no need of adding denaturant, easy amplification, good repeatability and good recovery rate.
5) Recombinant mHla-EpiVac proteins are capable of inducing animals to produce specific antibodies: subunit vaccine prepared by utilizing the recombinant mHla-EpiVac protein can be immunized by subcutaneous (intramuscular) injection, can excite organisms to generate high-titer IgG antibodies, and is proved to have good immunogenicity.
6) The recombinant mHla-EpiVac protein can induce specific humoral immunity and also can induce cellular immunity.
The klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, and the nucleotide sequence of the antigen protein is shown as SEQ ID NO. 1. Wherein mHla is a nontoxic mutant of staphylococcus aureus alpha hemolysin, and carrier protein (ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGT IAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN), serving as an epitope can form a heptamer; epiVac contains three immunodominant epitopes of klebsiella pneumoniae (KDNASNTQALSGGEIPGYDSTGR, NEDFNDTGKAAGLSDLSLKD, ADSKGNYASTGVSRSEHD) connected by a linker (GGGGS, GGGGS, GSGGSG). The invention also discloses a preparation method and application of the antigen protein. The antigen protein prepared by the method can effectively stimulate organisms to generate higher humoral immune response and cellular response, can provide obvious protection effect on killing dose of Klebsiella pneumoniae infection, and can be used as candidate antigens of Klebsiella pneumoniae vaccine.
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Drawings
The above and other features, properties and advantages of the present invention will become more apparent from the following description in conjunction with the accompanying drawings and embodiments.
Fig. 1: double enzyme digestion identification result of recombinant plasmid pGEX-6p-1-mHla-EpiVac
Lane M: the molecular weight standard (Marker) of nucleic acid (DNA) is as follows: 5000. 3000, 2000, 1500, 1000, 750, 500, 250, 100bp; lane 1: the identification result of recombinant expression plasmid pGEX-6p-1-mHla-EpiVac after double digestion by Nde1 and Xho1, the fragment separated after digestion is about 5900bp and about 1020bp; lane 2: plasmid pGEX-6p-1-mHla-EpiVac.
Fig. 2: mHla-EpiVac protein induced expression identification result
Lane M: protein molecular weight standard (Marker), size from top to bottom respectively: 180kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa; lane 1: a bacterial-destroying supernatant; lane 2: breaking bacteria and precipitating; lane 3: combining and flowing out; lane 4: filler bonding; lane 5: enzyme cutting of protein; lane 6: and (5) enzyme cutting of the filling material. The target protein is about 37kDa in size and is expressed in both supernatant and precipitate, but the protein has a large number of impurities and requires further purification.
Fig. 3: SDS-PAGE electrophoresis result of mHla-EpiVac protein after purification
Lane M: protein molecular weight standard (Marker), size from top to bottom respectively: 180kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa; lane 1: the mHla-EpiVac protein after cation exchange chromatography has better purity.
Fig. 4: specific IgG antibody titre to EpiVac after three immunizations
After three immunizations, specific IgG antibody titers against EpiVac were obtained, mHla-EpiVac immunization effectively increased antibody production, while EpiVac immunization produced little corresponding antibody.
Fig. 5A to 5C in fig. 5: spleen cell ELISPot after three immunizations
The specific cell number of IL-4 and IFN-gamma is measured, spleen cells of mice 7 days after three immunizations are stimulated again, and the result obtained by photographing and statistics after 40 hours of stimulation is proved to be effective cell immunity induction by experiments mHla-EpiVac.
Fig. 6A to 6C in fig. 6: protective Effect on mice after immunization mHla-EpiVac
Mice were given a lethal dose of YBQ x 106CFU lung infection 7 days after three immunizations, and then observed for ten days for survival, body weight, and clinically scored. Experiments show that the protection rate of mHla-EpiVac for single immunization can reach 50%, and the protection rate for the combination with an aluminum adjuvant can reach 80%, and the immunization effect of EpiVac is different from that of PBS; the body weight of the immunized mHla-EpiVac mice is less, the recovery is rapid, and the clinical scoring state is better.
Fig. 7: uptake of mHla-EpiVac by mice BMDCs
Immature mice BMDCs were incubated with mHla-EpiVac and EpiVac for 10h, respectively, and experiments demonstrated that immature mice BMDCs had more uptake of mHla-EpiVac and less uptake of EpiVac.
Detailed Description
The invention is further described below with reference to the drawings and examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The strains and various reagents used in the invention are as follows:
The plasmid pGEX-6p-1 and the escherichia coli strain BL21 are stored for the unit where the inventor is located, and restriction enzymes Nde I and Xho I and a protein Marker are large company TakaRa; plasmid extraction kits and gel recovery kits are products of Omega company, usa; cation exchange chromatography columns (HITRAPSP HP ml) and hydrophobic chromatography columns (HITRAP PHENYL HP ml) are products of company GE HEALTHCARE, U.S.A.
The inventors have studied and found 5 outer membrane proteins OMPs, ompW, fepA, ompK, ompA, ompK36, respectively, from KP outer membrane proteins by reverse vaccinology. The structure of the membrane is screened to obtain 22 loop structures and sequences thereof, serum of 7000721 standard strain infection recovery period is used for screening by ELISA method, and three high immunogenicity epitopes of FepA396-423, ompA148-165 and OmpW144-163 are obtained.
Step of ELISA screening for strongly immunogenic loop:
1) Diluting the purified 22 loop proteins to 5ug/mL by using coating liquid;
2) Coating: adding the recombinant protein diluent into an ELISA plate, 100 mu L/hole, washing 3 times with a washing solution after overnight at 4 ℃, wrapping with a preservative film after air drying, and placing in a refrigerator at 4 ℃ for standby;
3) Closing: adding 200 mu L/hole of sealing solution into the ELISA plate, placing the ELISA plate in an incubator at 37 ℃ for 2 hours, and washing for 3 times;
4) Serum from the third day after infection of the klebsiella pneumoniae 700721 strain is diluted 1:200;
5) Taking a sealed ELISA plate, sequentially adding diluted serum and 100 mu L/hole, placing the ELISA plate in an incubator at 37 ℃ for 1h, washing for 3 times, and air-drying;
6) HRP-labeled goat anti-mouse IgG antibody stock solution was diluted 1:10000, preparing an antibody working solution;
7) Adding diluted antibody working solution, 100 mu L/hole, placing in an incubator at 37 ℃ for 40min, washing for three times, and air drying;
8) Adding 100 mu L/hole of a substrate color development solution (TMB), and reacting for 5min at room temperature in a dark place;
9) Stop solution (2M H 2SO4) was added and immediately placed on an microplate reader to determine the OD at a wavelength of 450nm, screening for higher OD450 loop.
The 3 epitopes are fused and expressed as fusion antigen EpiVac, then mHla and EpiVac are fused to form antigen protein mHla-EpiVac, the antigen and aluminum adjuvant are combined to prepare vaccine immunized mice for three times, the toxicity attack protection rate of the high virulence strain YBQ screened in the early stage of a laboratory can reach 80%, the mHla-EpiVac can induce high-efficiency humoral immune response after the titer is measured, and ELISPot experiments prove that the protein can induce high-efficiency cellular immunity and can be used as effective vaccine candidate antigens.
Example 1: synthesis and subcloning of genes
1. Synthesis of the DNA sequence encoding mHla-EpiVac (SEQ ID NO: 1) and ligation of the sequence to pGEX-6p-1 was synthesized by Wuhan Jin Kairui Bioengineering Co.
2. Transformation of recombinant plasmid 1 tube E.coli BL21 competent cells (Shanghai Biotechnology Co., ltd.) were taken from a-80℃refrigerator and 4. Mu.l of the synthetic pGEX-6p-1-mHla-EpiVac plasmid was added. Ice bath for 30min, hot impact at 42 ℃ for 90s in metal bath, and rapid ice bath for 2min. 600 μl of LB blank medium was added, mixed well and placed in a shaking table at 37℃for 1h with shaking at 220 rpm. Mu.l of the bacterial liquid was applied to an ampicillin-resistant LB plate. The plates were placed in an incubator at 37℃for 14h. Well-separated colonies on the transformation plates were picked and inoculated into ampicillin-resistant LB medium and shake-cultured overnight at 37 ℃.
3. Double enzyme cutting identification
The plasmid of the positive clone was extracted by a rapid plasmid miniprep kit (Tiangen Biotechnology Co., ltd.) according to the procedure of the specification by shaking overnight at 37 ℃. Digestion was performed using Nde1 (Takara Co.) and Xhol (Takara Co.) in a water bath at 37℃for half an hour. The system is as follows:
A1.0% agarose gel was prepared by casting, wherein the gel contained EB (Shanghai Jun Cheng Biotechnology Co., ltd.) 0.5ug/ml, adding 1. Mu.l of 6X Loading buffer to each of the above cleavage reaction systems, and after electrophoresis for 20min by gel 80V, the cleavage results were observed by a UV scanner. As a result, the plasmid which had found positive clones was cut into 2 fragments, the large fragment of about 5900bp was the pGEX-6p-1 part of the expression vector, and the small fragment of about 1020bp was the inserted DNA fragment encoding mHla-EpiVac (FIG. 1).
Example 2: identification of KP-Ag1 antigen protein expression forms
Induction of expression by mHla-EpiVac
Taking 100 mu L of pGEX-6p-1-mHla-EpiVac/BL21 bacterial liquid cultured overnight, adding into 10mL Ampicillin resistant LB culture medium, culturing at 180rpm and 37 ℃ for 5 hours until the OD600 is 0.6-0.8, adding IPTG to the final concentration of 200 mu M, placing in a shaking table for induction expression, and inducing expression at 16 ℃ for 14 hours. Taking out the bacterial liquid after induction expression, centrifuging at 10000rpm for 5min, discarding the supernatant, adding 1mL PBS, mixing uniformly, performing ultrasonic lysis for 3min, centrifuging at 12000rpm at 4 ℃ for 15min, and separating the supernatant and the precipitate.
SDS-PAGE electrophoresis
Pouring 10% separating gel into offset plate, adding distilled water, flattening, standing at room temperature for 30min for solidification, pouring distilled water on upper layer, pouring concentrated gel, immediately inserting comb, standing at room temperature for 30min for solidification. And (5) taking 10 mu L of the treated samples respectively, and performing SDS-PAGE electrophoresis. The voltage is firstly 80v for 30min, then 180v is regulated, after electrophoresis for 1-2 h, the gel is taken out, put into transient blue staining solution for oscillation staining, put into primary water for oscillation decolorization, and then the result is observed under an imaging system, pGEX-6p-1-mHla-EpiVac/BL21 is expressed in a soluble form under the induction of the condition of 16 ℃, and a large amount of protein is also present in the sediment (figure 2).
Example 3: preparation of mHla-EpiVac protein antigen
1. Obtaining protein by amplifying culture
Taking 100 mu L of pGEX-6p-1-mHla-EpiVac/BL21 bacterial liquid stored in a refrigerator at 4 ℃ for standby, adding the bacterial liquid into 10mL of LB culture medium containing AMPICILLIN resistance for primary activation, culturing at 220rpm for 4-5h, taking 10mL of the primary activated bacterial liquid, adding the bacterial liquid into 2000mL of LB culture medium containing AMPICILLIN resistance for secondary activation, culturing at 37 ℃ for 4-5h until OD600 is 1.0, adding 400 mu L of IPTG (with the final concentration of 200 mu M), placing the bacterial liquid in a shaking table at 16 ℃ for 14h after induction, centrifuging at 10000rpm for 15min to collect bacterial bodies, adding 50mL of PBS (same as in example 2) for resuspension of bacterial bodies, and performing ultrasonic lysis on the bacterial liquid for 3min (200V), and collecting supernatant for subsequent purification.
Mhla-EpiVac protein purification
After washing Glutathione Sepharose mL of the affinity filler with 20mM PBS, pH7.5 for 3 times, the prepared supernatant was mixed with the affinity filler and combined at room temperature for 1 hour. Unbound supernatant was removed by air gravity column, the affinity packing was washed 3 times with PBS, and 1mg of PreScission protease was added in appropriate amount for excision of GST tag, and digested at room temperature for 2h. After the cleavage, the supernatant was collected for ion exchange chromatography.
Taking a cation exchange chromatographic column (HITRAPSP HP ml), adopting a PBS (same as in example 2) balance chromatographic system and an SP HP chromatographic column, loading the supernatant, adopting a wash buffer (50mM PB,pH 6.25,1M NaCl) to perform linear gradient elution, setting the elution flow rate to be 5ml/min, and setting the elution gradient to be 0-100% of the wash buffer and the elution volume to be 50ml. Collecting the eluted target protein and storing at 4 ℃ for standby. The result of electrophoresis is shown in FIG. 3.
Example 4: immunization and antibody detection of animals
Diluting mHla-EpiVac antigen with PBS, and adding Al (OH) 3 with concentration of 1mg/mL to prepare vaccine; BALB/C mice were immunized by double-sided thigh intramuscular injection with a 5-gauge half needle at days 0, 14 and 21, each with 100. Mu.L of antigen and 30. Mu.L of antigen, and the blank group was immunized with the same volume of PBS; on day 7 after the last immunization, tail vein blood of BALB/C mice was collected, and the antigen-specific IgG response level of the mice after immunization was detected by ELISA.
1. Preparation of liquid
1) Preparing a coating liquid: na 2CO31.6g,NaHCO3 2.9.9 g was weighed and dissolved in 1L ddH 2 O, and the pH was adjusted to 9.6 with a pH meter;
2) Preparing a sealing liquid: 1g of bovine serum albumin in 100mL of antibody diluent (1:100);
3) Preparation of antibody dilution: phosphate was dissolved in 1L ddH 2 O, 500. Mu.L Tween 20 was added, and the pH was adjusted to 7.4 with a pH meter;
4) Preparation of the washing liquid: co-antibody diluent
5) Color development liquid (TMB) is a product of Tiangen corporation;
6) Preparation of stop solution (2M H 2SO4): 22.2mL of concentrated sulfuric acid was poured into 177.8mL of ddH 2 O.
ELISA detection of antibody titers produced by mHla-EpiVac recombinant protein immunized mice
1) Diluting the purified mHla-EpiVac, fepA396-423, ompA148-165 and OmpW144-163 proteins to 5ug/mL with a coating solution;
2) Coating: adding the recombinant protein diluent into an ELISA plate, 100 mu L/hole, washing 3 times with a washing solution after overnight at 4 ℃, wrapping with a preservative film after air drying, and placing in a refrigerator at 4 ℃ for standby;
3) Closing: adding 200 mu L/hole of sealing solution into the ELISA plate, placing the ELISA plate in an incubator at 37 ℃ for 2 hours, and washing for 3 times;
4) Serum was diluted at 1:1000, 1:2000, 1:4000, 1:8000, etc.;
5) Taking a sealed ELISA plate, sequentially adding diluted serum and 100 mu L/hole, placing the ELISA plate in an incubator at 37 ℃ for 1h, washing for 3 times, and air-drying;
6) HRP-labeled goat anti-mouse IgG antibody stock solution was diluted 1:10000, preparing an antibody working solution;
7) Adding diluted antibody working solution, 100 mu L/hole, placing in an incubator at 37 ℃ for 40min, washing for three times, and air drying;
8) Adding 100 mu L/hole of a substrate color development solution (TMB), and reacting for 5min at room temperature in a dark place;
9) Adding a stop solution (2M H 2SO4), immediately placing on an enzyme-labeled instrument, and measuring an OD value at a wavelength of 450 nm;
10 Result judgment: a Sample of /A Negative of was positive (negative control was 1:1000 dilution of pre-mouse immune serum).
Results: the geometric average titer of EpiVac specific IgG antibody titer generated by mHla-EpiVac protein antigen immunized mice is 1:4096000; the geometric mean titer of EpiVac-specific IgG antibodies generated by the combination of mHla-EpiVac and aluminum adjuvant was 1:8192000 geometric mean titers against FepA396-423, ompA148-165, ompW144-16 specific IgG antibody titers were 1:8192000, respectively; 1:1024000;1:2048000; the antibody positive rate reaches 100% on the 7 th day after the last immunization (figure 4), which shows that mHla-EpiVac recombinant protein constructed by the invention has good immunogenicity.
Example 5: animal immunity and toxicity attack protection experiment
Diluting mHla-EpiVac antigen with PBS, and adding Al (OH) 3 with concentration of 1mg/mL to prepare vaccine; BALB/C mice were immunized by double-sided thigh intramuscular injection with a 5-gauge half needle at days 0, 14 and 21, each with 100. Mu.L of antigen and 30. Mu.L of antigen, and the blank group was immunized with the same volume of PBS; on day 7 after the last immunization, the mice are detoxified by adopting a mode of tracheal instillation by using 8X 10 6 CFU of klebsiella pneumoniae clinical strain YBQ, the survival state of the mice is observed, the observation period is 10 days, the death number of the mice is recorded every day, and the survival rate of the mice is calculated after the observation period is over. The results of three continuous animal experiments show that: compared with the control group, the survival rate of mHla-EpiVac immunized mice is obviously improved, the protection rate is about 50%, and the survival rate can reach 80% after the mice are combined with an aluminum adjuvant (fig. 5A-5C in fig. 5), so that the mice can be used as candidate antigens for vaccine development.
Example 6: ELISPot
Spleens of mice 7 days after three immunizations were taken, homogenized under sterile conditions, red-split using erythrocyte lysate, adjusted to a cell concentration of 1×10 7, and subsequently added to ELISpot plates (MabTech).
1) Each group of treatments:
(1) Activation of the pre-coated plates: firstly, 200uL of sterile PBS is added into each hole for washing, after standing for two minutes, the mixture is buckled out, and the process is repeated for 4 times; then 200. Mu.L 1640 complete medium was added to each well, and after 30 minutes of standing at room temperature, it was snapped out.
(2) Adding a cell suspension: the cell suspension was added to each well at a concentration of 100. Mu.L/well.
(3) Adding a stimulus: 100. Mu.L/well, specifically as follows:
Positive control wells: adding positive stimulant working solution.
Negative control wells: adding a medium (or a medium for resuspension of cells);
Experimental hole: mHla-EpiVac and EpiVac (formulated in serum-free medium or RPMI 1640) were added.
2) Color development
(1) The plate was emptied to remove cells and washed 5 times with 200 μl/well of PBS.
(2) The detection antibody (R4-6A 2-biotin) was diluted to 1. Mu.g/ml in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). 100 μl/well was added and incubated for 2 hours at room temperature
(3) 200 Μl/well of PBS was washed 5 times.
(4) Streptavidin HRP (1:1000) was diluted in PBS-0.5% FCS and 100 μl/well was added. Incubate for 1 hour at room temperature.
(5) 200 Μl/well of PBS was washed 5 times.
(6) 100 Μl/well of the ready-to-use TMB matrix solution was added and developed until a visible spot appeared.
(7) The color development was stopped by extensive washing in deionized water. The rack is removed from the plastic tray and the underside of the membrane is then rinsed.
(8) ELISPOT plate spot counts and records various parameters of spots for statistical analysis
Results: mHla-EpiVac is capable of inducing specific IL-4 and IFN-gamma cells, and further IL-4 and IFN-gamma secreting cells are generated by restimulation with mHla-EpiVac (FIGS. 6A-6C of FIG. 6), indicating that mHla-EpiVac is capable of generating specific cellular immunity after immunization.
Example 7: confocal laser
MHla-EpiVac and EpiVac were fluorescent according to the procedure of fluorescent dye AF488, fluorescent mHla-EpiVac and EpiVac were diluted at a concentration of 40. Mu.g/ml using 1640 medium containing 10% FBS, then incubated with 1X 10 5 of immature BMDCs mice in confocal dishes for 10h in the dark, medium was discarded, washed 3 times with 37℃pre-warmed PBS, and fixed with 4% paraformaldehyde for 15 min. Washing with PBS for 3 times, adding PBS containing 3% BSA and Triten-100, allowing to permeate for 15 min, discarding, and washing with PBS for 3 times; dyeing with 100nmol/l of phalloidin (absin) for 10 min, discarding, and washing with PBS for 3 times; staining with DAPI (bi yun tian) for 5min, discarding, and washing with PBS for 3 times; 100ul of PBS was added and the mixture was observed on the machine.
Results: uptake of mHla-EpiVac and EpiVac by immature BMDCs was greater for mHla-EpiVac at the same time and concentration (FIG. 7), indicating that mHla-EpiVac promotes uptake of antigen by immature BMDCs.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention; modifications and equivalent substitutions are intended to be included in the scope of the claims without departing from the spirit and scope of the present invention.
Claims (10)
1. A klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac is characterized by having an amino acid sequence shown in SEQ ID NO. 1.
2. The fusion antigen mHla-EpiVac of claim 1, wherein the fusion antigen comprises three immunodominant epitopes of klebsiella pneumoniae origin.
3. A fusion antigen mHla-EpiVac according to claim 2 wherein the amino acid sequences of the three immunodominant epitopes are FepA396-423 as shown in SEQ ID No.2, ompA148-165 as shown in SEQ ID No. 3, ompW144-163 as shown in SEQ ID No. 4.
4. The method for preparing the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of any of the preceding claims, comprising the steps of:
1) mHla-EpiVac synthesis and subcloning;
2) mHla-EpiVac;
3) mHla-EpiVac protein antigen is prepared;
4) mHla-EpiVac protein purification.
5. The method of claim 4, wherein step 1) comprises: mHla was ligated to FepA396-423 sequence shown in SEQ ID NO. 2 with a flexible strand, then ligated to OmpW144-163 sequence shown in SEQ ID NO. 4 with a flexible strand, and then ligated to OmpA148-165 sequence shown in SEQ ID NO. 3 with a flexible strand to form fusion antigen mHla-EpiVac.
6. The method of claim 4 or 5, wherein step 1) comprises constructing a recombinant expression plasmid using ampicillin-resistant prokaryotic expression plasmid pGEX-6 p-1.
7. The method according to any one of claims 4 to 6, wherein step 3) comprises amplifying the induced protein obtained in step 2).
8. The method of claim 7, wherein step 4) comprises: collecting genetically engineered bacteria expressing mHla-EpiVac; breaking bacteria according to high pressure and centrifuging; cation exchange; sequential combinations of hydrophobic chromatography purify mHla-EpiVac.
9. Use of the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of claim 1 in the preparation of an immune formulation for preventing or treating klebsiella pneumoniae.
10. A klebsiella pneumoniae injectable immune formulation comprising the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of claim 1, and any one or any combination of the following adjuvants: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete freund's adjuvant, incomplete freund's adjuvant, mycobacterial bcg adjuvant.
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