Truncated-form streptococcus hemolyticus bacteriolysin O and detection kit using same

A technology of hemolytic streptococcus and detection kits, applied in the direction of biological testing, application, material inspection products, etc., can solve the problems of low yield, affecting cell growth, etc., and achieve the effect of increasing yield

Active Publication Date: 2013-03-13
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The expression scheme reported in the prior art generally only removes the signal peptide part of the protein, and expresses most of the remaining protein, but because the toxicity of the remaining part affects cell growth, the yield is not high

Method used

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  • Truncated-form streptococcus hemolyticus bacteriolysin O and detection kit using same
  • Truncated-form streptococcus hemolyticus bacteriolysin O and detection kit using same
  • Truncated-form streptococcus hemolyticus bacteriolysin O and detection kit using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Cloning of truncated SLO gene and construction of expression vector

[0072] Streptococcus strains were inoculated into Meat Infusion Broth, and cultured with shaking at 37°C for 18-24 hours. The cells were collected by centrifugation, and the pellet was resuspended in 1 ml TE (pH 8.0). Add 6 μl of 50 mg / ml lysozyme, act at 37°C for 2 hours, then add 50 μl of 2mol / L NaCl, 110 μl of 10% SDS, 3 μl of 20 mg / ml proteinase K, and act at 50°C for 15 minutes. Afterwards, divide the bacterial liquid into 10ml centrifuge tubes, add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix well and let stand for 5min. Centrifuge at 12000rpm for 10min, and absorb the supernatant. Repeat the extraction twice. Add 0.6 times the volume of isopropanol to the supernatant, mix well, and let stand at room temperature for 10 min. Centrifuge at 12000rpm for 10min, wash the precipitate with 75% ethanol, dry it in air, and dissolve it in 50μl ddH 2 O middle.

[0073] Design p...

Embodiment 2

[0074] Expression of truncated SLO protein

[0075] Select SLO genetically engineered colonies from LB solid medium, inoculate them in LB medium containing 100ug / ml for overnight culture at 37°C, dilute them in LB medium at 1:1000 the next day, and cultivate them at 37°C until OD2.0, IPTG was added at a final concentration of 1mM for induction, and the bacterial fluid was harvested 4 hours later. The bacterial solution without IPTG was used as the uninduced control.

[0076] Take 500 μl of the bacterial solution before and after induction, remove the supernatant after centrifugation, resuspend the bacteria with 200 μl of protein loading buffer, boil for 5 minutes and centrifuge, and use the supernatant for SDS-PAGE detection. Coomassie Brilliant Blue staining results showed that after IPTG induction, there was a specific concentrated protein band at the predicted molecular weight, while the uninduced cells did not. For specific results, please refer to Figure 4 . The strai...

Embodiment 3

[0084] Preservation of engineered bacteria, expanded culture and purification of target protein

[0085] Bacteria storage method:

[0086] Pick the pET28b-SLO genetically engineered colony on the LB+Kan (100ug / ml) solid plate, inoculate it in the liquid medium of LB+kan (100ug / ml), and after the OD600 grows to about 2.0, store it until it contains glycerol The final concentration of glycerol in the frozen tube was 15%.

[0087] Cell activation and shake flask fermentation operation method:

[0088] Take the cryopreservation tube, after the bacterial solution is dissolved, streak culture (37°C) on LB+Kan (100ug / ml) solid plate, after a single colony grows, pick a single colony and inoculate it on LB+Kan (100ug / ml) ml) liquid medium, 37°C 150rpm / min overnight culture (about 12h), inoculated in LB+Kan (100ug / ml) liquid medium at 37°C 220rpm / min according to 0.1% inoculum size. When the culture medium grows to about OD600=1, add the inducer IPTG, the final concentration is 1mM,...

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Abstract

The invention provides a truncated-form streptococcus hemolyticus bacteriolysin O gene and an ASO detection kit. According to the invention, part of SLO gene fragments are cloned, and the part of SLO genes are cloned into an expression vector of escherichia coli and the like so as to realize mass expression and facilitate purification. In particular, the invention provides a DNA sequence which has a base sequence as shown in SEQ ID NO:1. According to the invention, the truncated-form SLO sequence can greatly improve the yield of SLO protein; more than 150 mg of SLO protein can be obtained through purification of one liter of bacterial culture, and the purity is more than 90%; immunological tests prove that the sectionally expressed SLO still maintains the specific immunogenicity.

Description

technical field [0001] The invention relates to a recombinant protein gene and a kit. More specifically, it relates to a truncated hemolytic streptolysin O recombinant protein and ASO detection kit. Background technique [0002] Hemolytic streptolysin O (Streptolysin O, SLO) is a secreted protein expressed by a variety of hemolytic streptococci. Humans will produce specific antibodies to SLO after infection with hemolytic streptococci. Antistreptolysin O (ASO) detection is one of the important indicators for clinical diagnosis of hemolytic streptococcal infection. The purified SLO is mainly derived from the purification of SLO in Streptococcus hemolyticus, and the expression and purification of SLO protein in Escherichia coli expression strains. Both of the above two methods have some limitations. Purification of SLO from hemolytic streptococcus has a low yield and is difficult to produce on a large scale. At the same time, there are hidden dangers in biological safety; th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/315C12N15/31C12N15/63G01N33/53
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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