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Hp tetravalent antigen as well as preparation method and application thereof

A technology of antigen and egg yolk antibody, applied in the field of biomedicine, can solve the problems of poor purity, low neutralizing activity, and poor effect of egg yolk antibody

Inactive Publication Date: 2020-10-20
南京蛋球球生物医学技术合伙企业(有限合伙)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The problems and shortcomings of the prior art are mainly that the prepared anti-Hp yolk antibody has low neutralizing activity to Hp, low antibody titer, large batch variation, lack of stability, and low extraction efficiency of specific antibodies, thus Reduced its effect of preventing and treating diseases caused by Hp infection
The main reasons for these problems include 1) the antigens used for immunization have low purity and poor immunogenicity, which leads to low antibody titers induced; 2) the anti-Hp yolk antibodies produced by a single antigen have poor effect on the prevention and treatment of Hp infection; 3 ) The process of extracting and purifying yolk antibody from eggs leads to poor purity of the extracted yolk antibody, which affects its neutralization and inhibition of Hp

Method used

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  • Hp tetravalent antigen as well as preparation method and application thereof
  • Hp tetravalent antigen as well as preparation method and application thereof
  • Hp tetravalent antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: a kind of Hp tetravalent antigen, it is urease element (UreB), vacuolar cytotoxin (VacA), cytotoxin gene A (CagA) and Hp flagellin sheath protein (HpaA) four kinds of antigen proteins After the corresponding nucleotides are optimized, they are cloned into pET28a, pET30 or pColdII prokaryotic expression vectors, and the antigens are expressed by prokaryotic expression bacteria BL21, Rosetta or OrigamiB;

[0052] The protein sequence 1 corresponding to the urease element (UreB) is:

[0053] MKKISRKEYVSMYGPTTGDKVRLGDTDLIAEVEHDYTIYGEELKFGGGKTLREGMSQSNNPSKEE LDLIITNALIVDYTGIYKADIGIKDGKIAGIGKGGNKDMQDGVKNNLSVGPATEALAGEGLIVTAG GIDTHIHFISPQQIPTAFASGVTTMIGGGTGPADGTNATTITPGRRNLKWMLRAAEEYSMNLGFLAK GNASNDASLADQIEAGAIGFKIHEDWGTTPSAINHALDVADKYDVQVAIHTDTLNEAGCVEDTMA AIAGRTMHTFHTEGAGGGHAPDIIKVAGEHNILPASTNPTIPFTVNTEAEHMDMLMVCHHLDKSIK EDVQFADSRIRPQTIAAEDTLHDMGIFSITSSDSQAMGRVGEVITRTWQTADKNKKEFGRLKEEKG DNDNFRIKRYLSKYTINPAIAHGISEYVGSVEVGKVADLVLWSPAFFGVKPNMIIKGGFIALSQMGD ANASIPTPQP...

Embodiment 2

[0063] Embodiment 2: The preparation method of Hp tetravalent antigen comprises the following steps:

[0064] (11) Prokaryotic bacterial culture: Streak inoculation of prokaryotic expression bacteria BL21, Rosetta or OrigamiB on LB culture plate, and put in 37°C incubator overnight. On the second day, pick a single colony and culture it overnight in LB medium at 37°C, 250rpm shaker; on the third day, inoculate the overnight cultured bacterial solution into fresh LB medium at 37°C, 4000 Rpm culture 1.5h to OD600 to 0.6. Transfer the bacterial culture solution into a centrifuge tube, place it on ice for 10 minutes, and centrifuge at 4000 rpm at 4°C for 10 minutes; discard the supernatant and replace it with pre-cooled 0.05M CaCl 2 Gently resuspend the cells in the solution, place on ice for 30 minutes, centrifuge at 4000rpm at 4°C for 10 minutes; discard the supernatant, add pre-cooled 0.05M CaCl containing 15% glycerol 2 solution, resuspended cells, placed on ice, aliquoted i...

Embodiment 3

[0068] Embodiment 3: the preparation method of the egg that contains the yolk antibody of Hp tetravalent antigen, comprises the following steps:

[0069] (21) emulsifying the Hp tetravalent antigen obtained through genetic engineering with an adjuvant; the adjuvant is Freund's complete adjuvant;

[0070] (22) Immune laying hens: select healthy laying hens aged 10 to 40 weeks and raise them in isolation for subcutaneous injection. Each hen is injected with 2-6 injection points. The amount of antigen used for immunization is 100 μg / chicken. The first immunization After that, booster immunization every two weeks;

[0071] (23) Collect the eggs produced by the last immunization, that is, obtain the eggs containing the egg yolk antibody of Hp tetravalent antigen.

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Abstract

The invention discloses an Hp tetravalent antigen, which is prepared by optimizing nucleotides corresponding to four antigen proteins of UreB, VacA, CagA and HpaA, cloning the optimized nucleotides into a pET28a, pET30 or pColdII prokaryotic expression vector, and carrying out prokaryotic expression by using prokaryotic expression bacteria BL21, Rosetta or OrigamiB to obtain the antigen. The Hp tetravalent antigen is used for preparing an egg with a yolk antibody containing the Hp tetravalent antigen, and an egg yolk antibody for efficiently blocking Hp colonization and infection; compared with an existing product, the yolk antibody has good solubility and high product purity, and the novel yolk antibody containing the tetravalent antigen Hp can block colonization of Hp, effectively inhibit the activity of Hp and efficiently prevent and treat gastrointestinal diseases caused by Hp infection. The Hp tetravalent antigen can be widely applied to manufacturing of food, food additives, medicines and health-care food.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a tetravalent antigen containing Hp and its preparation method and application. Background technique [0002] Helicobacter pylori (Hp) is a spiral-shaped, Gram-negative microaerophilic bacillus that requires harsh growth conditions. It is the only microbial species that can survive in the human stomach so far. Hp infection can lead to gastrointestinal diseases such as peptic ulcer, gastritis and gastric cancer, and is related to extragastric diseases such as coronary heart disease, diabetes and hepatobiliary disease. Epidemiology shows that 65%-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 my country, 15%-20% of patients with Hp infection develop peptic ulcer, 1% develop gastric cancer (gastric cancer, gastric mucosa-associated B-cell lymphoma (MALT lymphoma)). [00...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/12C07K16/02C07K1/36C07K1/34C07K1/22C12N15/62C12N15/70
CPCC07K14/195C07K16/02C07K16/1203C07K2317/11C07K2319/00C12N15/70C12N2800/22
Inventor 唐小军
Owner 南京蛋球球生物医学技术合伙企业(有限合伙)
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