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Preparation and application of anti-staphylococcus aureus polyclonal antibody

A polyclonal antibody, staphylococcus technology, applied in the fields of bioengineering and immunology, can solve problems such as unsatisfactory effect and accelerated drug-resistant Staphylococcus aureus

Inactive Publication Date: 2014-12-17
钱泓
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the combined use of antibiotics is mostly used in clinical treatment of Staphylococcus aureus infection, but the effect is not very satisfactory, and it also accelerates the emergence of drug-resistant Staphylococcus aureus

Method used

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  • Preparation and application of anti-staphylococcus aureus polyclonal antibody
  • Preparation and application of anti-staphylococcus aureus polyclonal antibody
  • Preparation and application of anti-staphylococcus aureus polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Construction of embodiment 1 expression vector pET28a-mntc

[0018] Using the clinically isolated cow Staphylococcus aureus genome as a template, the PCR results are as follows: figure 1 As shown, the nucleotide sequence of the obtained MntC gene is shown in SEQ ID NO.2.

[0019] 1.1 The sample loading system is (50μl):

[0020]

[0021]

[0022] 1.2 PCR amplification procedure:

[0023]

[0024] 1.3 Double digestion reaction (40μl system):

[0025]

[0026] Into a 1.5ml EP tube, add sample and mix according to step 1.3, and then place the two 40μl reaction solutions in a constant temperature water bath at 37°C for 2h.

[0027] 1.4 Gel recovery of DNA fragments:

[0028] (1) Perform 0.8% agarose gel electrophoresis (110 V for 30 min) on 40 μl of the system reaction solution in step 1.3;

[0029] (2) Under the ultraviolet light, cut the gel and recover the DNA fragments in a 1.5ml EP tube;

[0030] (3) Add 500 μl PC buffer to the 1.5ml EP tube in step (...

Embodiment 2

[0064] Example 2 Transformation of Escherichia coli BL21

[0065] Take 1 μl of plasmid and add it to 100 μl BL21 competent cells, and put it in ice bath for 30 minutes;

[0066] Heat shock at 42℃ for 90s;

[0067] Ice bath for 2 minutes;

[0068] Add 900 μl non-resistant LB culture solution in the ultra-clean bench;

[0069] Shake at 180rpm at 37°C for 1h;

[0070] Aspirate 100 μl of the bacterial solution and apply it to a Kana-resistant LB plate, and incubate overnight at 37°C.

Embodiment 3

[0071] Example 3 Large amount of induced expression

[0072] Bacteria picking: Pick a single clone into 50ml of kana-resistant LB culture medium and culture overnight at 37°C;

[0073] Transfer: transfer the bacterial solution to 500ml of kana-resistant LB culture medium at a ratio of 1:100, shake 3.5L in total, culture at 37°C and 220rpm for 2-2.5h to OD 600 value to 0.6;

[0074] Induction: bacterial solution OD 600 After the value reaches 0.6, add 500 μl IPTG (1M) to a final concentration of IPTG of 1 mmol / L, induce culture at 37°C and 220 rpm for 4 hours;

[0075] Bacteria collection: the bacteria solution was centrifuged at 6,000rpm for 10min to collect the bacteria; the bacteria were washed with 40ml PBS, centrifuged at 6,000rpm for 10min, the bacteria were collected, and stored at -20°C;

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Abstract

The invention discloses a novel anti-staphylococcus aureus polyclonal antibody for treating staphylococcus aureus infection. The antibody is obtained by immunizing a New Zealand white rabbit with staphylococcus aureus MntC protein as a major immunizing antigen. The novel anti-staphylococcus aureus polyclonal antibody has an immune regulation effect and is capable of promoting the phagocytosis of the organism to the staphylococcus aureus so as to achieve the effect of treating the staphylococcus aureus infection.

Description

technical field [0001] The invention relates to a staphylococcus aureus therapeutic polyclonal antibody, which belongs to the fields of bioengineering and immunology. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, referred to as SA), also known as "Staphylococcus aureus", is a Gram-positive coccus, often arranged in clusters of grapes, and can produce golden yellow pigment, and thus derived name. Staphylococcus aureus is one of the most important causes of nosocomial infection and acquired immune group infection. It can widely infect the body, including from skin inflammation, toxic shock, pneumonia to life-threatening osteomyelitis, endocarditis, sepsis, etc. a range of diseases. In addition, Staphylococcus aureus can also produce a variety of toxins that endanger human health. Staphylococcus aureus is ubiquitous in nature. In recent years, the US Centers for Disease Control reported that infections caused by Staphylococcus aureus accounted f...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K14/31C12N15/70A61K39/40A61P31/04
Inventor 钱泓吴有强覃思宣春玲车影曹珊珊张屹峰张丽丽吴素芳
Owner 钱泓
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