CN111793137A - Hp tetravalent antigen and preparation method and application thereof - Google Patents
Hp tetravalent antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Hp tetravalent antigen, which is characterized in that nucleotides corresponding to four antigen proteins of UreB, VacA, CagA and HpaA are optimized, then cloned into a pET28a, pET30 or pColdII prokaryotic expression vector, and antigens obtained by expression of prokaryotic expression bacteria BL21, Rosetta or origamB are utilized to prepare eggs containing yolk antibodies of the Hp tetravalent antigen and yolk antibodies capable of efficiently blocking the colonization and infection of Hp; compared with the existing product, the yolk antibody has good solubility and high product purity, and the novel yolk antibody containing tetravalent antigen Hp can block the permanent planting of Hp, effectively inhibit the activity of Hp, and can effectively prevent and treat gastrointestinal diseases caused by Hp infection. Can be widely applied to the manufacture of food, food additives, medicines and health-care food.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a tetravalent antigen containing Hp, and a preparation method and application thereof.
Background
Helicobacter pylori (Hp) is a spiral, gram-negative microaerophilic bacterium that is critical for growth conditions and is the only microbial species that has been found to be viable in the human stomach to date. Hp infection can cause gastrointestinal diseases such as peptic ulcer, gastritis and gastric cancer, and is related to diseases outside the stomach such as coronary heart disease, diabetes, liver and gallbladder diseases. Epidemiology has shown that 65% to 80% of gastric ulcers and more than 90% of duodenal ulcers are caused by Hp infection. And Hp infection is the main cause of gastric cancer in China, 15% -20% of patients infected by Hp suffer from peptic ulcer, and 1% suffer from gastric malignant tumor (gastric cancer, gastric mucosa-associated B cell lymphoma (MALT lymphoma)).
Hp is infected in people of different ethnicities and different regions, the global infection rate is about 50%, particularly in developing countries, the infection rate in China is 40-90%, and the average infection rate is 59%. And the infection age of China is earlier than that of developed countries by nearly 20 years, the infection rate of 20-40 years old is 45.4-63.6%, and the infection rate of more than 70 years old is up to 78.9%.
The helicobacter pylori bacillosis is mainly caused by that Hp destroys gastric cells, and the current experiments and theories prove that the helicobacter pylori bacillosis is mainly related to three proteins, namely uroase (Ure) expressed by Hp in vivo, vacuolar cytotoxin (VacA) and cytotoxin-associated gene A (cytotoxic-associated protein, CagA). Ure is an enzyme rich in helicobacter, can change the local strong acid environment in the stomach, plays an important role in the gastric parasitism and pathogenesis of the helicobacter, and all Hp bacteria discovered at present contain urease. UreB is an active center forming the Ure, has good conservation and antigenicity and is an ideal Hp vaccine target. VacA exists in the genome of all Hp strains, the expression product of the VacA is the only protein toxin secreted by Hp, and the VacA is an important virulence factor and can change cell membranes to form transmembrane anion channels, so that acid hydrolase is released to cause cell swelling, and epithelial cells are subjected to vacuole-like degeneration along with cell membrane fusion. CagA is one of Hp virulence markers and is related to Hp pathogenicity and clinical related disease severity.
Studies on Hp have shown that Hp colonizes the gastric mucosa by virtue of its specific binding of surface ligands to receptors for epithelial cells of the gastric mucosa. HpaA is a flagellum membrane protein of Hp, is one of important adhesins, exists on the surface of almost all clinically isolated Hp strains, and has the characteristics of surface exposure, high antigen conservation and the like. In addition, immunization of animals with the HpaA vaccine induces protective use.
The World Health Organization (WHO) has listed Hp as a class I carcinogen in 1994. With the continuous understanding of Hp, in 2015, global consensus on Kyoto clarified that Hp gastritis is a chronic infectious disease, it is suggested that patients infected with Hp should be eradicated unless there are counter factors (advanced age, basic disease, re-infection, etc.). However, the cure rate of the standard triple therapy (PPI + clarithromycin + adriamycin/metronidazole) recommended by global consensus at present is only about 70%, however, in recent years, 20-50% of Hp primary drug resistance rate of clarithromycin and 40-70% of metronidazole are reported in China, and Hp can generate double, triple or multiple drug resistance to the antibacterial drugs, and the double drug resistance rate of clarithromycin and metronidazole is more than 25%, so the scheme is not suitable for China any more. It is currently well recommended in the fifth national consensus that tetranection therapy with bismuth agents is recommended as the first choice. Bismuth agents can additionally increase eradication rates by 30-40% for Hp-resistant strains without drug resistance, thereby reducing overuse of antibiotics, but this approach is only suitable for short-term use. Therefore, the traditional four-combination therapy for treating Hp infection has low targeting property, antibiotics are easy to generate drug resistance, and the four-combination therapy has more side effects and high recurrence rate. Therefore, a safe and effective solution for resisting Hp infection is lacking in clinic and market at present.
Cholera enterotoxin (CT) is an important virulence factor of vibrio cholerae and contains two subunits, namely A and B. CTB removes the toxicity of CT, has mucosal adjuvant activity, can effectively stimulate the body to secrete antitoxin antibodies, and the most basic receptor GM1 exists on the surface of most mammalian cells, so the CTB can be used as a good adjuvant. CTB can 1) assist in enhancing the presentation of DC and other APC to antigen, and promote antigen to cross mucosal barrier; 2) inducing the organism to secrete specific antibodies; 3) stimulating the expression of receptors and the secretion of cytokines on the surface of T cells, B cells and APC. Many scholars at home and abroad successfully construct expression vectors of CTB and antigen or antigenic determinant fusion protein and express the CTB and the antigen or antigenic determinant fusion protein in host bacteria, so that the method for enhancing the immunogenicity of specific antigens through the fusion protein is a feasible way.
Domestic and foreign research shows that Hp-resisting specific yolk immunoglobulin (IgY) prepared with helicobacter pylori bacteria or some kind of pathogenic factor recombinant protein can reach Hp infection resisting effect and has no resistance. The Yolk Immunoglobulin (IgY) is the only Immunoglobulin in Yolk, and the IgY has the advantages of fast production, high yield, high titer, stable physicochemical property, wide source and no toxic or side effect. Anti-Hp-IgY can resist the degradation of pepsin in the stomach, and can generate immune response to Hp infection at the level of mucous membrane after entering the body, thereby reducing inflammation and ulcer caused by the Anti-Hp-IgY; can also specifically aim at Hp infection, can neutralize Hp protein toxin after being taken orally, block the toxin activity of the Hp protein toxin on epithelial cells, eradicate or lighten Hp infection and improve the immunity of human bodies.
The traditional treatment scheme has the following defects (as shown in figure 5) that the long-term use of PPI causes the Hp to have gastric antral to gastric metastasis, so that the risk of gastric cancer is increased; the bismuth agent can cause black stool and cause adverse reactions such as neurotoxicity and the like after long-term use; the use of antibiotics leads to an increase in drug-resistant strains, and abuse of antibiotics, in addition to increasing the drug-resistance rate, can also cause impairment of liver and kidney functions.
In the prior art, the total protein of a strain extracted by cracking Hp or a specific protein expressed by prokaryotic recombination, such as VacA protein, is used for immunizing laying hens according to a certain immunization program, then egg yolks are collected, and a yolk antibody for resisting Hp (such as VacA) is extracted by a water dilution method combined with an ammonium sulfate or PEG precipitation method, or is directly prepared into yolk powder for food additives or functional health products by freeze-drying.
The problems and the defects of the prior art are that the prepared anti-Hp yolk antibody has low Hp neutralization activity, low antibody titer, large batch difference and low stability, and the extraction efficiency of a specific antibody is low, so that the effect of preventing and treating diseases caused by Hp infection is reduced. The main reasons for these problems include 1) low purity of the antigen used for immunization, poor immunogenicity and thus low induced antibody titers; 2) the anti-Hp yolk antibody generated by a single antigen has poor effect on preventing and treating Hp infection; 3) the process problem of extracting and purifying the yolk antibody from the eggs leads to the low purity of the extracted yolk antibody and influences the neutralization and inhibition effect on Hp.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the existing problems, the yolk immunoglobulin is prepared by taking four antigens of urease, adhesin, vacuolating toxin and cytotoxicity related protein as immunogens, the antigens and CTB are fused and expressed to increase the immune effect of the antigens, the immune scheme and the purification method are improved to improve the yield and the purity of the yolk antibody, and the tetravalent Hp yolk antibody is obtained. The invention aims to provide a tetravalent antigen containing Hp, which has high antibody titer, stable quality, high extraction efficiency and good disease prevention effect, aiming at the defects of the prior art;
another object of the present invention is to provide a method for preparing Hp tetravalent antigen;
the third purpose of the invention is to provide a preparation method of eggs containing yolk antibody of Hp tetravalent antigen;
the fourth purpose of the invention is to provide an egg containing a yolk antibody of an Hp tetravalent antigen;
the fifth purpose of the invention is to provide a preparation method of the yolk antibody which can effectively block the Hp colonization and infection;
the sixth purpose of the invention is to provide a yolk antibody which can block the Hp colonization and infection with high efficiency;
the seventh object of the present invention is to provide the use of the yolk antibody capable of blocking the colonization and infection of Hp with high efficiency.
The technical scheme is as follows: the invention relates to a Hp tetravalent antigen, which is an antigen obtained by optimizing nucleotides corresponding to four antigen proteins, namely urozyme (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA) and flagellium membrane protein (HpaA) of Hp, cloning the optimized nucleotides into pET28a, pET30 or pColdII prokaryotic expression vectors and expressing the optimized nucleotides by prokaryotic expression bacteria BL21, Rosetta or OrigamiB;
the protein sequence 1 corresponding to the urozyme (UreB) is as follows:
MKKISRKEYVSMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEE LDLIITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAG GIDTHIHFISPQQIPTAFASGVTTMIGGGTGPADGTNATTITPGRRNLKWMLRAAEEYSMNLGFLAK GNASNDASLADQIEAGAIGFKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMA AIAGRTMHTFHTEGAGGGHAPDIIKVAGEHNILPASTNPTIPFTVNTEAEHMDMLMVCHHLDKSIK EDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGRLKEEKG DNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGFIALSQMGD ANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGLERQVLPVKNCRNITKKDM QFNDTTAHIEVNPETYHVFVDGKEVTSKPANKVSLAQLFSIFHHHHHHHH
the protein sequence 2 corresponding to the vacuolar cytotoxin (VacA) is:
HYWVKGGQWNKLEVDMKDAVGTYKLSGLINYTGGDLDVNMQKATLRLGQFNGNSFTSFKDSA DRTTRVDFNAKNISIDNFLEINNRVGSGAGRKASSTVLTLQASEGITSGKNAEISLYDGATLNLASNS VKLMGNVWMGRLQYVGAYLAPSYSTINTSKVTGEVNFNHLTVGDRNAAQAGIIASKKTYIGTLD LWQSAGLNIIAPPEGGYKDKPNNTTSQSGAKNDKNESAKNDKQDSN
the protein sequence 3 corresponding to the cytotoxin gene A (CagA) is as follows:
MTNEAINQQPQTEAAFNPQQFINNLQVAFIKVDNVVASFDPNQKPIVDKNDRDNRQAFEKISQLRE EFANKAIKNPTKKNQYFSSFISKSNDLIDKDNLIDTGSSIKSFQKFGTQRYQIFMNWVSHQNDPSKI NTQKIRGFMENIIQPPISDDKEKAEFLRSAKQAFAGIIIGNQIRSDQKFMGVFDESLKERQEAEKNGE PNGDPTGGDWLDIFLSFVFNKKQSSDLKETLNQEPVPHVQPDVATTTTDIQSLPPEARDLLDERGN FSKFTLGDMNMLDVEGVADIDPNYKFNQLLIHNNALSSVLMGSHNGIEPEKVSLLYGNNGGPEAR HDWNATVGYKNQRGDNVATLINVHMKNGSGLVIAGGEKGINNPSFYLYKEDQLTGSQRALSQEEI QNKVDFMEFLAQNNAKLDNLSKKEKEKFQNEIEDFQKDSKAYLDALGNDHIAFVSKKDKKHLAL VAEFGNGELSYTLKDYGKKADKALDREAKTTLQGSLKHDGVMFVDYSNFKYTNASKSPDKGVG ATNGVSHLEAGFSKVAVFNLPNLNNLAITSVVRQDLEDKLIAKGLSPQEANKLVKDFLSSNKELVG KALNFNKAVAEAKNTGNYDEVKQAQKDLEKSLKKRERLEKDVAKNLESKSGNKNKMEAKSQAN SHHHHHHHH
the protein sequence 4 corresponding to the flagellium membrane protein (HpaA) of Hp is as follows:
MKANNHFKDFAWKKCLLGASEVALLVGCSPHIIETNEVALKLNYHPASEKVQALDEKILLLRPAFQYSDNIAKEYENKFKNQTALKVEQILQNQGYKVITLDTSDKDDFSFSQKKEGYLALAMNAEIVLRPDPKRTI QKKSEPGLLFSTGLDKMEGVLIPAGFIKVTILEPMSGESLDSFTMDLSELDIQEKFLKTTHSSHSGGLVST MVKGTDNSNDAMKSALNKIFANIMQEIDKKLTQKNLESYQKDAKELKKKRNR。
the nucleotide optimization method specifically comprises the following steps: selecting codons of prokaryotic expression bacteria; protein tags to increase protein purification and detection; the CTB sequence is added through the N end or the C end of the antigen, so that the immune effect of the antigen is increased;
the sequence 5 corresponding to the CTB specifically is:
GTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN。
the invention also provides a preparation method of the Hp tetravalent antigen, which comprises the following steps:
(11) culturing prokaryotic bacteria: and (3) streaking prokaryotic expression bacteria BL21, Rosetta or OrigamiB onto an LB culture plate, and putting the LB culture plate in an incubator at 35-40 ℃ overnight. The next day, picking a single colony in an LB culture medium, and carrying out shaking table overnight culture at the temperature of 35-40 ℃ and the rpm of 200-300; and on the third day, inoculating the overnight cultured bacterial liquid into a fresh LB culture medium according to the ratio of 1:100, and culturing at the temperature of 35-40 ℃ and the rpm of 3000-5000 for 1-2 h until the OD600 is 600-0.6. Transferring the bacterial culture solution into a centrifugal tube, placing the centrifugal tube on ice for 5-15 minutes, and centrifuging the centrifugal tube at 4 ℃ and 3000-5000 rpm for 10 minutes; discard the supernatant and use pre-cooled 0.05M CaCl2The solution is used for gently resuspending cells, is placed on ice for 30 minutes, and is centrifuged at 3000-5000 rpm at 4 ℃ for 10 minutes; the supernatant was discarded and pre-cooled 0.05M CaCl containing 15% glycerol was added2The solution is used for resuspending cells, is placed on ice, is subpackaged by 100-200 mu l per tube, is quick-frozen by liquid nitrogen, and is preserved at the temperature of-70 ℃;
(12) transforming plasmids containing flagellin of urozyme, vacuolar cytotoxin, cytotoxin gene A and Hp into prokaryotic expression bacteria: taking out the sensitive state of the prokaryotic expression bacteria in the step (1) from a refrigerator at the temperature of-70 ℃, unfreezing the sensitive state at room temperature, and standing the unfrozen sensitive state on ice; respectively adding the 50ng of plasmid into a competence; performing water bath heat shock at 40-45 ℃ for 80-100 seconds, and then placing on ice for 2 minutes; adding 1ml LB to culture, and carrying out shaking culture at 35-40 ℃ and 150rpm for 40-50 minutes; 100 mul of the bacterial liquid is taken and coated on an agarose culture plate containing corresponding resistance;
(13) prokaryotic expression of tetravalent antigen: selecting a single colony on a culture plate, adding 10ml of LB culture medium, culturing at 35-40 ℃ and 200-300 rpm overnight, inoculating the colony to an LB culture medium containing corresponding resistance at a ratio of 1: 100-1: 500, continuing to culture, adding IPTG (isopropyl thiogalactoside) for induction expression when OD600 is 1.0, and continuing to culture for 2-3 hours; centrifuging at 4 ℃ for 10 minutes at 3000-5000 rpm, removing supernatant, adding a proper amount of PBS (phosphate buffer solution) for resuspension, adding PMSF (permanent magnet phosphate) until the final concentration is 0.1mM, and precooling on ice for 10 minutes; carrying out ultrasonic crushing for 10-30 minutes until bacterial liquid is clarified; centrifuging at 4 ℃ and 12000rpm for 10 minutes, and taking the supernatant; detecting protein expression by SDS-PAGE electrophoresis;
(14) purification of tetravalent antigen: purifying the ultrasonic supernatant bacterial liquid by an affinity column, treating the purified protein solution by a desalting column, and performing ultrafiltration concentration on the obtained protein solution to obtain the Hp tetravalent antigen.
Further, the affinity column in step (14) is a nickel column.
The invention also provides a preparation method of the egg containing the yolk antibody of the Hp tetravalent antigen, which comprises the following steps:
(21) emulsifying the Hp tetravalent antigen obtained by genetic engineering by adopting an adjuvant;
(22) immunizing egg-laying hens: selecting healthy laying hens of 10-40 weeks old for isolated feeding, performing subcutaneous injection, selecting 2-6 injection points for each hen for injection, and strengthening the immunity once every two weeks after the first immunization, wherein the antigen amount for immunization is 100 mu g/chicken;
(23) collecting the eggs laid by the last immunization to obtain the eggs containing the yolk antibody of the Hp tetravalent antigen. Further, the adjuvant in the step (21) is Freund's complete adjuvant, Freund's incomplete adjuvant or aluminum hydroxide adjuvant. The invention also provides the eggs containing the yolk antibody of the Hp tetravalent antigen prepared by the scheme.
The invention also provides a preparation method of the yolk antibody containing the Hp tetravalent antigen, which separates and purifies eggs of the yolk antibody containing the Hp tetravalent antigen to obtain the yolk antibody containing the Hp tetravalent antigen, and comprises the following specific steps: cleaning and sterilizing egg yolk antibodies containing tetravalent antigens Hp, and opening the opening to allow egg white to flow out; turning and rolling the yolk on common filter paper for several times, removing egg white, and breaking yolk membrane to collect yolk; diluting the yolk with sterile PBS (phosphate buffer solution) with the volume of 1-3 times of that of the yolk, stirring at room temperature, slowly adding 40% PEG6000 solution until the final concentration is 3-4%, and continuously stirring for 20-50 minutes; centrifuging at 8000-15000 rpm at 4 ℃ for 10 minutes, collecting the upper solution after centrifugation, and filtering by filter paper to remove insoluble substances; continuously adding 40% PEG6000 solution into the supernatant until the final concentration is 9.5%, stirring at room temperature for 20min, and centrifuging at 8000-15000 rpm for 10 min at 4 ℃; dissolving the precipitate with PBS (yolk volume) and adding 40% PEG6000 to final concentration of 12%, stirring at room temperature for 30 min, centrifuging under the same conditions, removing supernatant, and re-suspending the precipitate with 1/2 PBS (yolk volume); adding saturated ammonium sulfate solution at 4 deg.C under stirring, stirring for 1 hr, centrifuging under the same conditions, and removing supernatant; the sediment is resuspended and dissolved by PBS with the yolk volume of 1/10-1/20; dialyzing the dissolved egg yolk antibody for 20 hours by using PBS (phosphate buffer solution), replacing dialysate every 4-6 hours, and collecting the dialyzed egg yolk antibody; collecting the dialyzed yolk antibody, diluting by 50-100 times, and measuring the absorbance at 280nm to calculate the content of the yolk antibody; the purity of the obtained yolk antibody is identified by SDS-PAGE electrophoresis.
The invention also provides a yolk antibody containing the Hp tetravalent antigen prepared by the method.
The invention also provides the application of the yolk antibody containing the tetravalent antigen Hp in the preparation of food, food additives, medicines and health-care food.
Has the advantages that: (1) according to the invention, the prepared Hp tetravalent antigen is prepared by a special preparation method, and the Hp tetravalent antigen is applied to prepare the yolk antibody containing the tetravalent antigen Hp, the yolk antibody has good solubility and higher product purity compared with the existing product, the novel yolk antibody containing the tetravalent antigen Hp can block the permanent planting of Hp, effectively inhibit the activity of Hp, and can efficiently prevent and treat gastrointestinal diseases caused by Hp infection; (2) compared with the prior art, the method for extracting and purifying the yolk antibody from the eggs can greatly improve the purity of the extracted yolk antibody and improve the neutralization and inhibition effects of the extracted yolk antibody on Hp.
Drawings
FIG. 1 is a graph comparing the dissolution of the yolk antibody containing the tetravalent antigen Hp of the present invention with the existing products in the market;
FIG. 2 is a graph comparing the absorbance values of the supernatant after dissolution of the yolk antibody containing the tetravalent antigen Hp of the present invention and the commercial products;
FIG. 3 is a graph showing the comparison of the IgY content of the products of the present invention containing the yolk antibody with the tetravalent antigen Hp with the products currently available in the market by Western blotting;
FIG. 4 is a graph showing the comparison between the relative expression levels of a product containing a yolk antibody with a tetravalent antigen Hp and a product existing in the market by analyzing Western blotting bands using ImageJ software.
FIG. 5 traditional four-combination anti-Hp therapy and its drawbacks.
Detailed Description
The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the embodiments.
Example 1: a Hp tetravalent antigen is an antigen obtained by optimizing nucleotides corresponding to four antigen proteins, namely urokinase (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA) and flagellium membrane protein (HpaA) of Hp, cloning the optimized nucleotides into pET28a, pET30 or pColdII prokaryotic expression vectors and expressing the optimized nucleotides by prokaryotic expression bacteria BL21, Rosetta or origamB;
the protein sequence 1 corresponding to the urozyme (UreB) is as follows:
MKKISRKEYVSMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEE LDLIITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAG GIDTHIHFISPQQIPTAFASGVTTMIGGGTGPADGTNATTITPGRRNLKWMLRAAEEYSMNLGFLAK GNASNDASLADQIEAGAIGFKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMA AIAGRTMHTFHTEGAGGGHAPDIIKVAGEHNILPASTNPTIPFTVNTEAEHMDMLMVCHHLDKSIK EDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGRLKEEKG DNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGFIALSQMGD ANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGLERQVLPVKNCRNITKKDM QFNDTTAHIEVNPETYHVFVDGKEVTSKPANKVSLAQLFSIFHHHHHHHH
the protein sequence 2 corresponding to the vacuolar cytotoxin (VacA) is:
HYWVKGGQWNKLEVDMKDAVGTYKLSGLINYTGGDLDVNMQKATLRLGQFNGNSFTSFKDSA DRTTRVDFNAKNISIDNFLEINNRVGSGAGRKASSTVLTLQASEGITSGKNAEISLYDGATLNLASNS VKLMGNVWMGRLQYVGAYLAPSYSTINTSKVTGEVNFNHLTVGDRNAAQAGIIASKKTYIGTLD LWQSAGLNIIAPPEGGYKDKPNNTTSQSGAKNDKNESAKNDKQDSN
the protein sequence 3 corresponding to the cytotoxin gene A (CagA) is as follows:
MTNEAINQQPQTEAAFNPQQFINNLQVAFIKVDNVVASFDPNQKPIVDKNDRDNRQAFEKISQLRE EFANKAIKNPTKKNQYFSSFISKSNDLIDKDNLIDTGSSIKSFQKFGTQRYQIFMNWVSHQNDPSKI NTQKIRGFMENIIQPPISDDKEKAEFLRSAKQAFAGIIIGNQIRSDQKFMGVFDESLKERQEAEKNGE PNGDPTGGDWLDIFLSFVFNKKQSSDLKETLNQEPVPHVQPDVATTTTDIQSLPPEARDLLDERGN FSKFTLGDMNMLDVEGVADIDPNYKFNQLLIHNNALSSVLMGSHNGIEPEKVSLLYGNNGGPEAR HDWNATVGYKNQRGDNVATLINVHMKNGSGLVIAGGEKGINNPSFYLYKEDQLTGSQRALSQEEI QNKVDFMEFLAQNNAKLDNLSKKEKEKFQNEIEDFQKDSKAYLDALGNDHIAFVSKKDKKHLAL VAEFGNGELSYTLKDYGKKADKALDREAKTTLQGSLKHDGVMFVDYSNFKYTNASKSPDKGVG ATNGVSHLEAGFSKVAVFNLPNLNNLAITSVVRQDLEDKLIAKGLSPQEANKLVKDFLSSNKELVG KALNFNKAVAEAKNTGNYDEVKQAQKDLEKSLKKRERLEKDVAKNLESKSGNKNKMEAKSQAN SHHHHHHHH
the protein sequence 4 corresponding to the flagellium membrane protein (HpaA) of Hp is as follows:
MKANNHFKDFAWKKCLLGASEVALLVGCSPHIIETNEVALKLNYHPASEKVQALDEKILLLRPAFQYSDNIAKEYENKFKNQTALKVEQILQNQGYKVITLDTSDKDDFSFSQKKEGYLALAMNAEIVLRPDPKRTI QKKSEPGLLFSTGLDKMEGVLIPAGFIKVTILEPMSGESLDSFTMDLSELDIQEKFLKTTHSSHSGGLVST MVKGTDNSNDAMKSALNKIFANIMQEIDKKLTQKNLESYQKDAKELKKKRNR。
the nucleotide optimization method specifically comprises the following steps: selecting codons of prokaryotic expression bacteria; protein tags to increase protein purification and detection; the CTB sequence is added through the N end or the C end of the antigen, so that the immune effect of the antigen is increased;
the sequence 5 corresponding to the CTB specifically is:
GTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN。
example 2: the preparation method of the Hp tetravalent antigen comprises the following steps:
(11) culturing prokaryotic bacteria: prokaryotic expression bacteria BL21, Rosetta or OrigamiB were streaked onto LB plates and placed in a 37 ℃ incubator overnight. On the next day, picking a single colony in an LB culture medium, and shaking at 37 ℃ and 250rpm for overnight culture; on the third day, the overnight cultured broth was inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37 ℃ and 4000rpm for 1.5h to an OD600 of 0.6. Transferring the bacterial culture solution into a centrifugal tube, placing the centrifugal tube on ice for 10 minutes, and centrifuging the centrifugal tube at the temperature of 4 ℃ and the rpm of 4000 for 10 minutes; discard the supernatant and use pre-cooled 0.05M CaCl2The solution was gently resuspended in cells, placed on ice for 30 minutes, and centrifuged at 4000rpm at 4 ℃ for 10 minutes; the supernatant was discarded and pre-cooled 0.05M CaCl containing 15% glycerol was added2The solution is used for resuspending cells, is placed on ice, is subpackaged by 100 mu l per tube, is quick-frozen by liquid nitrogen, and is preserved at the temperature of minus 70 ℃;
(12) transforming plasmids containing flagellin of urozyme, vacuolar cytotoxin, cytotoxin gene A and Hp into prokaryotic expression bacteria: taking out the sensitive state of the prokaryotic expression bacteria in the step (1) from a refrigerator at the temperature of-70 ℃, unfreezing the sensitive state at room temperature, and standing the unfrozen sensitive state on ice; respectively adding the 50ng of plasmid into a competence; hot shocking in 42 ℃ water bath for 90 seconds, and then placing on ice for 2 minutes; adding 1ml LB culture, 37 ℃, 150rpm shaking culture 45 minutes; 100 mul of the bacterial liquid is taken and coated on an agarose culture plate containing corresponding resistance;
(13) prokaryotic expression of tetravalent antigen: selecting a single colony on a culture plate, adding 10ml of LB culture medium for culture at 37 ℃ and 250rpm overnight, inoculating the colony into an LB culture medium containing corresponding resistance at a ratio of 1:100, continuing to culture, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression when OD600 is between 1.0, and continuing to culture for 2.5 hours; centrifuging at 4 ℃ and 4000rpm for 10 minutes, removing supernatant, adding a proper amount of PBS (phosphate buffer solution) for resuspension, adding PMSF (permanent magnet phosphate) until the final concentration is 0.1mM, and precooling on ice for 10 minutes; carrying out ultrasonic crushing for 25 minutes until bacterial liquid is clear; centrifuging at 4 ℃ and 12000rpm for 10 minutes, and taking the supernatant; detecting protein expression by SDS-PAGE electrophoresis;
(14) purification of tetravalent antigen: purifying the ultrasonic supernatant bacterial liquid by a nickel column, treating the purified protein solution by a desalting column, and performing ultrafiltration concentration on the obtained protein solution to obtain the Hp tetravalent antigen.
Example 3: the preparation method of the egg containing the yolk antibody of the Hp tetravalent antigen comprises the following steps:
(21) emulsifying the Hp tetravalent antigen obtained by genetic engineering by adopting an adjuvant; the adjuvant is Freund's complete adjuvant;
(22) immunizing egg-laying hens: selecting healthy laying hens of 10-40 weeks old for isolated feeding, performing subcutaneous injection, selecting 2-6 injection points for each hen for injection, and strengthening the immunity once every two weeks after the first immunization, wherein the antigen amount for immunization is 100 mu g/chicken;
(23) collecting the eggs laid by the last immunization to obtain the eggs containing the yolk antibody of the Hp tetravalent antigen.
Example 5: the preparation method of the yolk antibody containing the Hp tetravalent antigen comprises the following steps of separating and purifying eggs of the yolk antibody containing the Hp tetravalent antigen to obtain the yolk antibody containing the Hp tetravalent antigen: cleaning and sterilizing egg yolk antibodies containing tetravalent antigens Hp, and opening the opening to allow egg white to flow out; turning and rolling the yolk on common filter paper for several times, removing egg white, and breaking yolk membrane to collect yolk; diluting yolk with sterile PBS (2 times of yolk volume), stirring at room temperature, slowly adding 40% PEG6000 solution to final concentration of 3.5%, and stirring for 30 min; centrifuging at 4 deg.C at 10000rpm for 10 min, collecting upper layer solution, and filtering with filter paper to remove insoluble substances; adding 40% PEG6000 solution into the supernatant to final concentration of 9.5%, stirring at room temperature for 20min, centrifuging at 4 deg.C and 10000rpm for 10 min; dissolving the precipitate with PBS (yolk volume) and adding 40% PEG6000 to final concentration of 12%, stirring at room temperature for 30 min, centrifuging under the same conditions, removing supernatant, and re-suspending the precipitate with 1/2 PBS (yolk volume); adding saturated ammonium sulfate solution at 4 deg.C under stirring, stirring for 1 hr, centrifuging under the same conditions, and removing supernatant; the pellet was resuspended and solubilized with 1/10 egg yolk volume of PBS; dialyzing the dissolved egg yolk antibody for 20 hours by using PBS (phosphate buffer solution), replacing dialysate every 4-6 hours, and collecting the dialyzed egg yolk antibody; collecting dialyzed yolk antibody, diluting by 100 times, measuring absorbance at 280nm, and calculating the content of the yolk antibody; the purity of the obtained yolk antibody is identified by SDS-PAGE electrophoresis.
The yolk antibody containing the tetravalent antigen Hp prepared by the invention can be applied to the manufacture of food, food additives, medicines and health-care food.
Example 6: the yolk antibody of 2 g Hp tetravalent antigen is added into 100 g milk powder to obtain food, which can be used for adjuvant treatment of Hp infected patient or for protecting digestive tract mucosa.
Example 7: mixing yolk antibody of Hp tetravalent antigen with Hericium erinaceus polysaccharide at a ratio of 1:4, and encapsulating to obtain health product for adjuvant treatment of Hp infection patients or protection of digestive tract mucosa.
Example 8: mixing yolk antibody of Hp tetravalent antigen and spirulina powder at a ratio of 1:6, adding adjuvants, tabletting, and making into oral tablet for adjuvant treatment of Hp infection patients or for protecting digestive tract mucosa.
Compared with the existing products in the market, the yolk antibody containing the Hp tetravalent antigen prepared by the scheme of the invention has the advantages of good solubility and higher purity. In the following experiments, the product containing IgY is named as the rotten and quiet chewable tablet, produced by Shandong Rui Ying pharmaceutical group GmbH; the egg yolk powder is sold as egg yolk powder and produced by Jiangsu kang de egg industry Co.
Experiment I, the yolk antibody (tetravalent anti-Hp-IgY) containing Hp tetravalent antigen provided by the invention has better solubility
The experimental method comprises the following steps: weighing tetravalent anti-Hp-IgY, and preparing drugs with the concentrations of 1mg/mL, 10mg/mL, 100mg/mL, 200mg/mL and 500 mg/mL; certain brand egg yolk powder and certain IgY-containing commercial products are respectively weighed to prepare the concentrations of 1mg/mL, 10mg/mLl and 100 mg/mL. The drug dissolution was observed. The absorbance values were measured at a wavelength of 600nm using a microplate reader.
The experimental results are as follows: the tetravalent anti-Hp-IgY has better solubility and good solubility, and is easy to dissolve in distilled water or PBS. As shown in FIG. 1, it can be seen from the experimental phenomenon that 0.5g of tetravalent anti-Hp-IgY can be completely dissolved in 1mL of water without visible suspended matter and precipitate, 0.1g of a commercial product containing IgY can be added into 1mL of water to show obvious suspended matter and precipitate, 0.01g of a brand egg yolk powder can be added into 1mL of water to show obvious precipitate, and the solubility is poor. When the tetravalent anti-Hp-IgY, a certain brand of yolk powder and a certain IgY-containing commodity are singly compared at the concentration of 10mg/ml, the tetravalent anti-Hp-IgY is obviously and completely dissolved, no visible particle suspension and precipitation exists, the certain IgY-containing commodity is also completely dissolved, the auxiliary material or the auxiliary material can make the solution be light orange, and the suspended matter and precipitation are obviously and possibly insoluble. All solutions were tested for absorbance at a wavelength of 600nm using a microplate reader and plotted using Prism 5 software, as shown in FIG. 2. The solvent, namely distilled water, is taken as a reference, the absorbance values are compared, the interference of the self color of the medicine or the auxiliary material is removed, and the dissolubility of the IgY is obviously better when the absorbance of the quadrivalent anti-Hp-IgY and the absorbance of a certain IgY-containing commodity and a certain brand of yolk powder are compared.
Experiment II, the tetravalent anti-Hp (Hp-IgY) provided by the invention has higher content
The experimental method comprises the following steps: westernblotting determination of protein content in samples (1) preparation of protein samples of known protein concentration (tetravalent anti-Hp-IgY, a certain IgY-containing commodity and a certain brand of egg yolk powder) were mixed with 5 × loadingbuffer according to a volume ratio of 1:4, denatured by a metal bath at 95 ℃ for 5min, and stored in a refrigerator at-80 ℃ for later use. (the space structure of the protein is opened and the buffer is fully combined with the protein) (2) glue preparation. And preparing the separation gel and the concentrated gel according to the gel preparation kit specification. (preparation for subsequent gel electrophoresis) (3) loading. Cleaning a glass plate (avoiding the influence of experiment residual reagent on the experiment), detecting leakage, filling glue, transferring the glue preparation plate to an electrophoresis tank after the concentrated glue is solidified, taking down a comb after the electrophoresis buffer is filled, adding a denatured protein sample into a sample loading hole, and paying attention to ensure that the total protein content of each hole is consistent. A marker is added in another hole. (molecular weight reference) (4) electrophoresis. Adding a proper amount of electrophoresis buffer solution into the electrophoresis box, assembling an electrophoresis apparatus, carrying out electrophoresis at a constant voltage of 80V, and stopping electrophoresis when the sample runs to the bottom of the gel. (separation of different types of proteins in protein samples) (5) membrane transfer. After electrophoresis, the whole piece of gel is taken down, placed on a PVDF membrane activated by methanol, clamped by a membrane transferring clamp, placed in an electrophoresis box filled with membrane transferring buffer solution, and placed in an appropriate amount of ice bags. After the instrument was assembled, it was placed on ice and the membrane was spun at 106V for 90 min. (transferring the protein to a solid phase carrier to make the protein more stable and firm and facilitate subsequent experiments) (6). And after the membrane conversion is finished, putting the PVDF membrane into an appropriate amount of 5% skimmed milk powder prepared by TBST for membrane sealing, and then putting the PVDF membrane into an appropriate amount of 5% skimmed milk powder prepared by TBST for sealing for 2-4 hours. (seal other wells on PVDF membrane to avoid antibody entry into the well to generate false positives) (7) antibody incubation. After blocking was complete, the PVDF membrane was washed twice with the appropriate amount of TBST. The incubation was then performed with HRP-labeled anti-IgY antibody (abcam, ab 6877). Four hours at room temperature or 12-16 hours at 4 ℃. (IgY on the solid phase carrier is used as antigen to react with the corresponding antibody) and (8) is developed and imaged. And after the incubation of the antibody is finished, the antibody is collected again and stored in a refrigerator at the temperature of-80 ℃ for the next use. After washing the PVDF membrane three times with TBST, color observation was performed with ECL developing solution and a chemiluminescence developing apparatus, and the picture was preserved. (detection of specific positions of proteins bound to the antibody after electrophoretic separation) (9) Gray scale analysis. And (4) carrying out gray level analysis on the developed and stored picture by using image J software, and comparing the content of the IgY in the sample.
(for the determination of the IgY content).
From the experimental results of FIGS. 3 and 4, it was shown that the content of IgY of tetravalent IgY was higher than that of the commercial IgY-containing products and egg yolk powder.
As noted above, while the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limited thereto. Various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Nanjing egg ball biomedical technology partnership (limited partnership)
<120> Hp tetravalent antigen and preparation method and application thereof
<141>2019-12-12
<160>5
<170>SIPOSequenceListing 1.0
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Met Lys Lys Ile Ser Arg Lys Glu Tyr Val Ser Met Tyr Gly Pro Thr
1 5 10 15
Thr Gly Asp Lys Val Arg Leu Gly Asp Thr Asp Leu Ile Ala Glu Val
20 25 30
Glu His Asp Tyr Thr Ile Tyr Gly Glu Glu Leu Lys Phe Gly Gly Gly
35 40 45
Lys Thr Leu Arg Glu Gly Met Ser Gln Ser Asn Asn Pro Ser Lys Glu
50 55 60
Glu Leu Asp Leu Ile Ile Thr Asn Ala Leu Ile Val Asp Tyr Thr Gly
65 70 75 80
Ile Tyr Lys Ala Asp Ile Gly Ile Lys Asp Gly Lys Ile Ala Gly Ile
85 90 95
Gly Lys Gly Gly Asn Lys Asp Met Gln Asp Gly Val Lys Asn Asn Leu
100 105 110
Ser Val Gly Pro Ala Thr Glu Ala Leu Ala Gly Glu Gly Leu Ile Val
115 120 125
Thr Ala Gly Gly Ile Asp Thr His Ile His Phe Ile Ser Pro Gln Gln
130 135 140
Ile Pro Thr Ala Phe Ala Ser Gly Val Thr Thr Met Ile Gly Gly Gly
145 150 155 160
Thr Gly Pro Ala Asp Gly Thr Asn Ala Thr Thr Ile Thr Pro Gly Arg
165 170 175
Arg Asn Leu Lys Trp Met Leu Arg Ala Ala Glu Glu Tyr Ser Met Asn
180 185 190
Leu Gly Phe Leu Ala Lys Gly Asn Ala Ser Asn Asp Ala Ser Leu Ala
195 200 205
Asp Gln Ile Glu Ala Gly Ala Ile Gly Phe Lys Ile His Glu Asp Trp
210215 220
Gly Thr Thr Pro Ser Ala Ile Asn His Ala Leu Asp Val Ala Asp Lys
225 230 235 240
Tyr Asp Val Gln Val Ala Ile His Thr Asp Thr Leu Asn Glu Ala Gly
245 250 255
Cys Val Glu Asp Thr Met Ala Ala Ile Ala Gly Arg Thr Met His Thr
260 265 270
Phe His Thr Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Ile Lys
275 280 285
Val Ala Gly Glu His Asn Ile Leu Pro Ala Ser Thr Asn Pro Thr Ile
290 295 300
Pro Phe Thr Val Asn Thr Glu Ala Glu His Met Asp Met Leu Met Val
305 310 315 320
Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp
325 330 335
Ser Arg Ile Arg Pro Gln Thr Ile Ala Ala Glu Asp Thr Leu His Asp
340 345 350
Met Gly Ile Phe Ser Ile Thr Ser Ser Asp Ser Gln Ala Met Gly Arg
355 360 365
Val Gly Glu Val Ile Thr Arg Thr Trp Gln Thr Ala Asp Lys Asn Lys
370375 380
Lys Glu Phe Gly Arg Leu Lys Glu Glu Lys Gly Asp Asn Asp Asn Phe
385 390 395 400
Arg Ile Lys Arg Tyr Leu Ser Lys Tyr Thr Ile Asn Pro Ala Ile Ala
405 410 415
His Gly Ile Ser Glu Tyr Val Gly Ser Val Glu Val Gly Lys Val Ala
420 425 430
Asp Leu Val Leu Trp Ser Pro Ala Phe Phe Gly Val Lys Pro Asn Met
435 440 445
Ile Ile Lys Gly Gly Phe Ile Ala Leu Ser Gln Met Gly Asp Ala Asn
450 455 460
Ala Ser Ile Pro Thr Pro Gln Pro Val Tyr Tyr Arg Glu Met Phe Ala
465 470 475 480
His His Gly Lys Ala Lys Tyr Asp Ala Asn Ile Thr Phe Val Ser Gln
485 490 495
Ala Ala Tyr Asp Lys Gly Ile Lys Glu Glu Leu Gly Leu Glu Arg Gln
500 505 510
Val Leu Pro Val Lys Asn Cys Arg Asn Ile Thr Lys Lys Asp Met Gln
515 520 525
Phe Asn Asp Thr Thr Ala His Ile Glu Val Asn Pro Glu Thr Tyr His
530 535 540
Val Phe Val Asp Gly Lys Glu Val Thr Ser Lys Pro Ala Asn Lys Val
545 550 555 560
Ser Leu Ala Gln Leu Phe Ser Ile Phe His His His His His His His
565 570 575
His
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His Tyr Trp Val Lys Gly Gly Gln Trp Asn Lys Leu Glu Val Asp Met
1 5 10 15
Lys Asp Ala Val Gly Thr Tyr Lys Leu Ser Gly Leu Ile Asn Tyr Thr
20 25 30
Gly Gly Asp Leu Asp Val Asn Met Gln Lys Ala Thr Leu Arg Leu Gly
35 40 45
Gln Phe Asn Gly Asn Ser Phe Thr Ser Phe Lys Asp Ser Ala Asp Arg
50 55 60
Thr Thr Arg Val Asp Phe Asn Ala Lys Asn Ile Ser Ile Asp Asn Phe
65 70 75 80
Leu Glu Ile Asn Asn Arg Val Gly Ser Gly Ala Gly Arg Lys Ala Ser
85 90 95
Ser Thr Val Leu Thr Leu Gln Ala Ser Glu Gly Ile Thr Ser Gly Lys
100 105 110
Asn Ala Glu Ile Ser Leu Tyr Asp Gly Ala Thr Leu Asn Leu Ala Ser
115 120 125
Asn Ser Val Lys Leu Met Gly Asn Val Trp Met Gly Arg Leu Gln Tyr
130 135 140
Val Gly Ala Tyr Leu Ala Pro Ser Tyr Ser Thr Ile Asn Thr Ser Lys
145 150 155 160
Val Thr Gly Glu Val Asn Phe Asn His Leu Thr Val Gly Asp Arg Asn
165 170 175
Ala Ala Gln Ala Gly Ile Ile Ala Ser Lys Lys Thr Tyr Ile Gly Thr
180 185 190
Leu Asp Leu Trp Gln Ser Ala Gly Leu Asn Ile Ile Ala Pro Pro Glu
195 200 205
Gly Gly Tyr Lys Asp Lys Pro Asn Asn Thr Thr Ser Gln Ser Gly Ala
210 215 220
Lys Asn Asp Lys Asn Glu Ser Ala Lys Asn Asp Lys Gln Asp Ser Asn
225 230 235 240
<210>3
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Met Thr Asn Glu Ala Ile Asn Gln Gln Pro Gln Thr Glu Ala Ala Phe
1 5 10 15
Asn Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Ile Lys Val
20 25 30
Asp Asn Val Val Ala Ser Phe Asp Pro Asn Gln Lys Pro Ile Val Asp
35 40 45
Lys Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Lys Ile Ser Gln Leu
50 55 60
Arg Glu Glu Phe Ala Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn
65 70 75 80
Gln Tyr Phe Ser Ser Phe Ile Ser Lys Ser Asn Asp Leu Ile Asp Lys
85 90 95
Asp Asn Leu Ile Asp Thr Gly Ser Ser Ile Lys Ser Phe Gln Lys Phe
100 105 110
Gly Thr Gln Arg Tyr Gln Ile Phe Met Asn Trp Val Ser His Gln Asn
115 120 125
Asp Pro Ser Lys Ile Asn Thr Gln Lys Ile Arg Gly Phe Met Glu Asn
130 135 140
Ile Ile Gln Pro Pro Ile Ser Asp Asp Lys Glu Lys Ala Glu Phe Leu
145 150155 160
Arg Ser Ala Lys Gln Ala Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile
165 170 175
Arg Ser Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu
180 185 190
Arg Gln Glu Ala Glu Lys Asn Gly Glu Pro Asn Gly Asp Pro Thr Gly
195 200 205
Gly Asp Trp Leu Asp Ile Phe Leu Ser Phe Val Phe Asn Lys Lys Gln
210 215 220
Ser Ser Asp Leu Lys Glu Thr Leu Asn Gln Glu Pro Val Pro His Val
225 230 235 240
Gln Pro Asp Val Ala Thr Thr Thr Thr Asp Ile Gln Ser Leu Pro Pro
245 250 255
Glu Ala Arg Asp Leu Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr
260 265 270
Leu Gly Asp Met Asn Met Leu Asp Val Glu Gly Val Ala Asp Ile Asp
275 280 285
Pro Asn Tyr Lys Phe Asn Gln Leu Leu Ile His Asn Asn Ala Leu Ser
290 295 300
Ser Val Leu Met Gly Ser His Asn Gly Ile Glu Pro Glu Lys Val Ser
305 310315 320
Leu Leu Tyr Gly Asn Asn Gly Gly Pro Glu Ala Arg His Asp Trp Asn
325 330 335
Ala Thr Val Gly Tyr Lys Asn Gln Arg Gly Asp Asn Val Ala Thr Leu
340 345 350
Ile Asn Val His Met Lys Asn Gly Ser Gly Leu Val Ile Ala Gly Gly
355 360 365
Glu Lys Gly Ile Asn Asn Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gln
370 375 380
Leu Thr Gly Ser Gln Arg Ala Leu Ser Gln Glu Glu Ile Gln Asn Lys
385 390 395 400
Val Asp Phe Met Glu Phe Leu Ala Gln Asn Asn Ala Lys Leu Asp Asn
405 410 415
Leu Ser Lys Lys Glu Lys Glu Lys Phe Gln Asn Glu Ile Glu Asp Phe
420 425 430
Gln Lys Asp Ser Lys Ala Tyr Leu Asp Ala Leu Gly Asn Asp His Ile
435 440 445
Ala Phe Val Ser Lys Lys Asp Lys Lys His Leu Ala Leu Val Ala Glu
450 455 460
Phe Gly Asn Gly Glu Leu Ser Tyr Thr Leu Lys Asp Tyr Gly Lys Lys
465 470 475480
Ala Asp Lys Ala Leu Asp Arg Glu Ala Lys Thr Thr Leu Gln Gly Ser
485 490 495
Leu Lys His Asp Gly Val Met Phe Val Asp Tyr Ser Asn Phe Lys Tyr
500 505 510
Thr Asn Ala Ser Lys Ser Pro Asp Lys Gly Val Gly Ala Thr Asn Gly
515 520 525
Val Ser His Leu Glu Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu
530 535 540
Pro Asn Leu Asn Asn Leu Ala Ile Thr Ser Val Val Arg Gln Asp Leu
545 550 555 560
Glu Asp Lys Leu Ile Ala Lys Gly Leu Ser Pro Gln Glu Ala Asn Lys
565 570 575
Leu Val Lys Asp Phe Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala
580 585 590
Leu Asn Phe Asn Lys Ala Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr
595 600 605
Asp Glu Val Lys Gln Ala Gln Lys Asp Leu Glu Lys Ser Leu Lys Lys
610 615 620
Arg Glu Arg Leu Glu Lys Asp Val Ala Lys Asn Leu Glu Ser Lys Ser
625 630 635640
Gly Asn Lys Asn Lys Met Glu Ala Lys Ser Gln Ala Asn Ser His His
645 650 655
His His His His His His
660
<210>4
<211>260
<212>PRT
<213>Artificial Sequence
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Met Lys Ala Asn Asn His Phe Lys Asp Phe Ala Trp Lys Lys Cys Leu
1 5 10 15
Leu Gly Ala Ser Glu Val Ala Leu Leu Val Gly Cys Ser Pro His Ile
20 25 30
Ile Glu Thr Asn Glu Val Ala Leu Lys Leu Asn Tyr His Pro Ala Ser
35 40 45
Glu Lys Val Gln Ala Leu Asp Glu Lys Ile Leu Leu Leu Arg Pro Ala
50 55 60
Phe Gln Tyr Ser Asp Asn Ile Ala Lys Glu Tyr Glu Asn Lys Phe Lys
65 70 75 80
Asn Gln Thr Ala Leu Lys Val Glu Gln Ile Leu Gln Asn Gln Gly Tyr
85 90 95
Lys Val Ile Thr Leu Asp Thr Ser Asp Lys Asp Asp Phe Ser Phe Ser
100105 110
Gln Lys Lys Glu Gly Tyr Leu Ala Leu Ala Met Asn Ala Glu Ile Val
115 120 125
Leu Arg Pro Asp Pro Lys Arg Thr Ile Gln Lys Lys Ser Glu Pro Gly
130 135 140
Leu Leu Phe Ser Thr Gly Leu Asp Lys Met Glu Gly Val Leu Ile Pro
145 150 155 160
Ala Gly Phe Ile Lys Val Thr Ile Leu Glu Pro Met Ser Gly Glu Ser
165 170 175
Leu Asp Ser Phe Thr Met Asp Leu Ser Glu Leu Asp Ile Gln Glu Lys
180 185 190
Phe Leu Lys Thr Thr His Ser Ser His Ser Gly Gly Leu Val Ser Thr
195 200 205
Met Val Lys Gly Thr Asp Asn Ser Asn Asp Ala Met Lys Ser Ala Leu
210 215 220
Asn Lys Ile Phe Ala Asn Ile Met Gln Glu Ile Asp Lys Lys Leu Thr
225 230 235 240
Gln Lys Asn Leu Glu Ser Tyr Gln Lys Asp Ala Lys Glu Leu Lys Lys
245 250 255
Lys Arg Asn Arg
260
<210>5
<211>104
<212>PRT
<213>Artificial Sequence
<400>5
Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr
1 5 10 15
Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu
20 25 30
Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr
35 40 45
Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys
50 55 60
Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu
65 70 75 80
Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala
85 90 95
Ile Ala Ala Ile Ser Met Ala Asn
100
Claims (10)
1. An Hp tetravalent antigen characterized by: the method comprises the steps of optimizing nucleotides corresponding to four antigen proteins of urokinase, vacuolar cytotoxin, flagellum and tunica vaginalis protein of cytotoxin gene A and Hp, cloning the optimized nucleotides into pET28a, pET30 or pColdII prokaryotic expression vectors, and expressing the optimized nucleotides by prokaryotic expression bacteria BL21, Rosetta or OrigamiB to obtain antigens;
the protein sequence 1 corresponding to the urozyme is as follows:
MKKISRKEYVSMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEELDLIITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAGGIDTHIHFISPQQIPTAFASGVTTMIGGGTGPADGTNATTITPGRRNLKWMLRAAEEYSMNLGFLAKGNASNDASLADQIEAGAIGFKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMAAIAGRTMHTFHTEGAGGGHAPDIIKVAGEHNILPASTNPTIPFTVNTEAEHMDMLMVCHHLDKSIKEDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGRLKEEKGDNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGFIALSQMGDANASIPTPQPVYYREMFAHHGKAKYDANITFVSQAAYDKGIKEELGLERQVLPVKNCRNITKKDMQFNDTTAHIEVNPETYHVFVDGKEVTSKPANKVSLAQLFSIFHHHHHHHH
the protein sequence 2 corresponding to the vacuolar cytotoxin is as follows:
HYWVKGGQWNKLEVDMKDAVGTYKLSGLINYTGGDLDVNMQKATLRLGQFNGNSFTSFKDSADRTTRVDFNAKNISIDNFLEINNRVGSGAGRKASSTVLTLQASEGITSGKNAEISLYDGATLNLASNSVKLMGNVWMGRLQYVGAYLAPSYSTINTSKVTGEVNFNHLTVGDRNAAQAGIIASKKTYIGTLDLWQSAGLNIIAPPEGGYKDKPNNTTSQSGAKNDKNESAKNDKQDSN
the protein sequence 3 corresponding to the cytotoxin gene A is as follows:
MTNEAINQQPQTEAAFNPQQFINNLQVAFIKVDNVVASFDPNQKPIVDKNDRDNRQAFEKISQLREEFANKAIKNPTKKNQYFSSFISKSNDLIDKDNLIDTGSSIKSFQKFGTQRYQIFMNWVSHQNDPSKINTQKIRGFMENIIQPPISDDKEKAEFLRSAKQAFAGIIIGNQIRSDQKFMGVFDESLKERQEAEKNGEPNGDPTGGDWLDIFLSFVFNKKQSSDLKETLNQEPVPHVQPDVATTTTDIQSLPPEARDLLDERGNFSKFTLGDMNMLDVEGVADIDPNYKFNQLLIHNNALSSVLMGSHNGIEPEKVSLLYGNNGGPEARHDWNATVGYKNQRGDNVATLINVHMKNGSGLVIAGGEKGINNPSFYLYKEDQLTGSQRALSQEEIQNKVDFMEFLAQNNAKLDNLSKKEKEKFQNEIEDFQKDSKAYLDALGNDHIAFVSKKDKKHLALVAEFGNGELSYTLKDYGKKADKALDREAKTTLQGSLKHDGVMFVDYSNFKYTNASKSPDKGVGATNGVSHLEAGFSKVAVFNLPNLNNLAITSVVRQDLEDKLIAKGLSPQEANKLVKDFLSSNKELVGKALNFNKAVAEAKNTGNYDEVKQAQKDLEKSLKKRERLEKDVAKNLESKSGNKNKMEAKSQANSHHHHHHHH
the protein sequence 4 corresponding to the flagellium protein of Hp is as follows:
MKANNHFKDFAWKKCLLGASEVALLVGCSPHIIETNEVALKLNYHPASEKVQALDEKILLLRPAFQYSDNIAKEYENKFKNQTALKVEQILQNQGYKVITLDTSDKDDFSFSQKKEGYLALAMNAEIVLRPDPKRTIQKKSEPGLLFSTGLDKMEGVLIPAGFIKVTILEPMSGESLDSFTMDLSELDIQEKFLKTTHSSHSGGLVSTMVKGTDNSNDAMKSALNKIFANIMQEIDKKLTQKNLESYQKDAKELKKKRNR。
2. the Hp tetravalent antigen of claim 1, wherein: the nucleotide optimization method specifically comprises the following steps: selecting codons of prokaryotic expression bacteria; protein tags to increase protein purification and detection; the CTB sequence is added through the N end or the C end of the antigen, so that the immune effect of the antigen is increased;
the sequence 5 corresponding to the CTB specifically is:
GTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN。
3. a method for producing the Hp tetravalent antigen of claim 1, comprising the steps of:
(11) culturing prokaryotic bacteria: and (3) streaking prokaryotic expression bacteria BL21, Rosetta or OrigamiB onto an LB culture plate, and putting the LB culture plate in an incubator at 35-40 ℃ overnight. The next day, picking a single colony in an LB culture medium, and carrying out shaking table overnight culture at the temperature of 35-40 ℃ and the rpm of 200-300; and on the third day, inoculating the overnight cultured bacterial liquid into a fresh LB culture medium according to the ratio of 1:100, and culturing at the temperature of 35-40 ℃ and the rpm of 3000-5000 for 1-2 h until the OD600 is 600-0.6. Transferring the bacterial culture solution into a centrifugal tube, and placing for 5-15 minutes on iceCentrifuging at 3000-5000 rpm at 4 ℃ for 10 minutes; discard the supernatant and use pre-cooled 0.05M CaCl2The solution is used for gently resuspending cells, is placed on ice for 30 minutes, and is centrifuged at 3000-5000 rpm at 4 ℃ for 10 minutes; the supernatant was discarded and pre-cooled 0.05M CaCl containing 15% glycerol was added2The solution is used for resuspending cells, is placed on ice, is subpackaged by 100-200 mu l per tube, is quick-frozen by liquid nitrogen, and is preserved at the temperature of-70 ℃;
(12) transforming plasmids containing flagellin of urozyme, vacuolar cytotoxin, cytotoxin gene A and Hp into prokaryotic expression bacteria: taking out the sensitive state of the prokaryotic expression bacteria in the step (1) from a refrigerator at the temperature of-70 ℃, unfreezing the sensitive state at room temperature, and standing the unfrozen sensitive state on ice; respectively adding the 50ng of plasmid into a competence; performing water bath heat shock at 40-45 ℃ for 80-100 seconds, and then placing on ice for 2 minutes; adding 1ml LB to culture, and carrying out shaking culture at 35-40 ℃ and 150rpm for 40-50 minutes; 100 mul of the bacterial liquid is taken and coated on an agarose culture plate containing corresponding resistance;
(13) prokaryotic expression of tetravalent antigen: selecting a single colony on a culture plate, adding 10ml of LB culture medium, culturing at 35-40 ℃ and 200-300 rpm overnight, inoculating the colony to an LB culture medium containing corresponding resistance at a ratio of 1: 100-1: 500, continuing to culture, adding IPTG (isopropyl thiogalactoside) for induction expression when OD600 is 1.0, and continuing to culture for 2-3 hours; centrifuging at 4 ℃ for 10 minutes at 3000-5000 rpm, removing supernatant, adding a proper amount of PBS (phosphate buffer solution) for resuspension, adding PMSF (permanent magnet phosphate) until the final concentration is 0.1mM, and precooling on ice for 10 minutes; carrying out ultrasonic crushing for 10-30 minutes until bacterial liquid is clarified; centrifuging at 4 ℃ and 12000rpm for 10 minutes, and taking the supernatant; detecting protein expression by SDS-PAGE electrophoresis;
(14) purification of tetravalent antigen: purifying the ultrasonic supernatant bacterial liquid by an affinity column, treating the purified protein solution by a desalting column, and performing ultrafiltration concentration on the obtained protein solution to obtain the Hp tetravalent antigen.
4. The method for producing an Hp tetravalent antigen of claim 3, wherein: the affinity column in the step (14) is a nickel column.
5. A preparation method of eggs containing yolk antibodies of Hp tetravalent antigens is characterized by comprising the following steps:
(21) emulsifying the Hp tetravalent antigen obtained by genetic engineering by adopting an adjuvant;
(22) immunizing egg-laying hens: selecting healthy laying hens of 10-40 weeks old for isolated feeding, performing subcutaneous injection, selecting 2-6 injection points for each hen for injection, and strengthening the immunity once every two weeks after the first immunization, wherein the antigen amount for immunization is 100 mu g/chicken;
(23) collecting the eggs laid by the last immunization to obtain the eggs containing the yolk antibody of the Hp tetravalent antigen.
6. The method for producing an egg with a yolk antibody against a tetravalent antigen of Hp according to claim 5, wherein: the adjuvant in the step (21) is Freund's complete adjuvant, Freund's incomplete adjuvant or aluminum hydroxide adjuvant.
7. An egg having a yolk antibody against Hp tetravalent antigen prepared according to the protocol of claim 5.
8. A preparation method of a yolk antibody containing Hp tetravalent antigen is characterized in that: separating and purifying eggs containing the yolk antibody of the Hp tetravalent antigen to obtain the yolk antibody containing the Hp tetravalent antigen, which comprises the following steps: cleaning and sterilizing egg yolk antibodies containing tetravalent antigens Hp, and opening the opening to allow egg white to flow out; turning and rolling the yolk on common filter paper for several times, removing egg white, and breaking yolk membrane to collect yolk; diluting the yolk with sterile PBS (phosphate buffer solution) with the volume of 1-3 times of that of the yolk, stirring at room temperature, slowly adding 40% PEG6000 solution until the final concentration is 3-4%, and continuously stirring for 20-50 minutes; centrifuging at 8000-15000 rpm at 4 ℃ for 10 minutes, collecting the upper solution after centrifugation, and filtering by filter paper to remove insoluble substances; continuously adding 40% PEG6000 solution into the supernatant until the final concentration is 9.5%, stirring at room temperature for 20min, and centrifuging at 8000-15000 rpm for 10 min at 4 ℃; dissolving the precipitate with PBS (yolk volume) and adding 40% PEG6000 to final concentration of 12%, stirring at room temperature for 30 min, centrifuging under the same conditions, removing supernatant, and re-suspending the precipitate with 1/2 PBS (yolk volume); adding saturated ammonium sulfate solution at 4 deg.C under stirring, stirring for 1 hr, centrifuging under the same conditions, and removing supernatant; the sediment is resuspended and dissolved by PBS with the yolk volume of 1/10-1/20; dialyzing the dissolved egg yolk antibody for 20 hours by using PBS (phosphate buffer solution), replacing dialysate every 4-6 hours, and collecting the dialyzed egg yolk antibody; collecting the dialyzed yolk antibody, diluting by 50-100 times, and measuring the absorbance at 280nm to calculate the content of the yolk antibody; the purity of the obtained yolk antibody is identified by SDS-PAGE electrophoresis.
9. A yolk antibody containing Hp tetravalent antigen prepared by the method of claim 8.
10. Use of the yolk antibody containing the Hp tetravalent antigen of claim 9 in the manufacture of food, food additives, medicines and health foods.
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