Helicobacter pylori virulence hypotype mixed representative strain group and immunized cow milk prepared from same
A technology for Helicobacter pylori and Helicobacter pylori, applied in the biological field, can solve problems such as easy infection and inability to cover Helicobacter pylori, and achieve the effects of low cost, no drug resistance, and good patient compliance
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Embodiment 1
[0063] Example 1 Isolation, cultivation and identification of gastric Helicobacter pylori
[0064] The isolated population came from a total of 498 cases of Helicobacter pylori infection in Shijiazhuang area. Bite the gastric mucosal tissue of Helicobacter pylori infected patients through gastroscope, and then put the gastric mucosal tissue into a glass grinder containing 0.5ml of Brooke's broth and grind it into a slurry, draw 100 microliters and spread it on the Columbia selection medium Plate (containing 8% defibrillated sheep blood, 5mg / L TMP, 5mg / L polymyxin B, 5mg / L amphotericin B, 10mg / L vancomycin), and then place the plate in a three-gas culture Cultivated in the box (10% CO 2 , 85% N 2 , 5% O 2 ), cultured at 37°C for 3-7 days, and carried out Gram staining microscopic examination, urease, catalase, and oxidase experiments on the suspicious colonies cultured. The result is Gram-negative, S-type or arc-type, and the positive three-enzyme test can be initially iden...
Embodiment 2
[0065] Example 2 Gene PCR method of Helicobacter pylori
[0066] (1) Bacterial DNA extraction method
[0067] 1. Scrape a fresh bacterial lawn (soybean size) from the Helicobacter pylori medium and place it on the wall of a 1.5ml EP tube. Add 0.9% normal saline, shake and mix well, centrifuge at 10000rpm for 2 min, discard the supernatant, and repeat this 3 times;
[0068] 2. Add 180ul ATL (to lyse the bacteria and dissolve the precipitate);
[0069] 3. Add 20ul of proteinase K, vortex to mix, put in a 56-degree metal bath at 300rpm for 1-3 hours, and centrifuge briefly;
[0070] 4. Add 200ul AL, shake and mix, 70 degree metal bath for 10mins, and centrifuge instantly;
[0071] 5. Add 200ul of absolute ethanol, gently mix up and down until clarified for 15 seconds, and centrifuge briefly;
[0072] 6. Add 500ul AW1, centrifuge at 8000rpm for 1min, discard the tube and keep the column, and replace with a new collection tube;
[0073] 7. Add 500ul AW2, centrifuge at 14000rpm...
Embodiment 3
[0085] Example 3 Preparation of Dairy Cow Vaccine by Helicobacter pylori Virulence Subtype Mixed Representative Strain Group
[0086] (A) Helicobacter pylori infected patients were divided into A, B, and O groups according to the ABO blood group system, and Helicobacter pylori was isolated from the stomachs of patients infected with different blood types of Helicobacter pylori;
[0087] (B) PCR and sequencing of cagA, cagE, vacA, iceA, babA2 of Helicobacter pylori isolated from each blood type
[0088] (C) After sequencing cagA, cagE, vacA, iceA, babA2, according to blood type and gene combination: genotype cagA was screened from group A Helicobacter pylori + / cagE2 / s1a / m2 / i1 / iceA1, genotype cagA was screened from Helicobacter pylori group B + / cagE2 / s1a / m1b / i1 / iceA1, genotype cagA was screened from Helicobacter pylori group O + / cagE2 / s1a / m2 / babA2 / i1 / iceA1 three representative strains;
[0089] (D) Three popular representative strains of virulent subtypes were collected by...
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