Method for improving gene knockdown conversion rate of talaromyces marneffei

A Marneffei and gene knockout technology, applied in the biological field, can solve the problems of difficult preservation, morphological imbalance and rupture, long time consumption, etc., and achieves the effects of convenient preparation, reduced damage and improved transformation efficiency.

Inactive Publication Date: 2019-10-01
THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about the pathogenic mechanism of T. marneffei at home and abroad
[0003] Gene knockout technology is the basis for studying the infection mechanism, immune escape mechanism and gene function of T. marneffei. The cell wall of T. marneffei is hard to digest; The medium is easily broken and difficult to survive, etc., resulting in low transformation efficiency of this method. How to improve the preparation efficiency of T. marneffei protoplasts in a short period of time and how to promote the survival of the gene transformation has b...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for improving gene knockdown conversion rate of talaromyces marneffei
  • Method for improving gene knockdown conversion rate of talaromyces marneffei
  • Method for improving gene knockdown conversion rate of talaromyces marneffei

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0024] The present invention will be further described below.

[0025] The SD culture liquid base of the present invention refers to: 2% glucose medium; the described SD+U medium refers to: 2% glucose medium+uracil; the described OSMO refers to: 10mM potassium phosphate buffer ; The STC refers to: 1M sorbitol buffer; the PTC refers to: 60% polyethylene glycol buffer; the ANM-U solid medium refers to: basal medium.

[0026] A method for improving the gene knockout transformation rate of T. marneffei according to the present invention is carried out according to the following steps:

[0027] 1. Preparation of T. marneffei cells: Transfer 1 mL of T. marneffei spore liquid to 400 mL of SD+U medium, shake at 200 rpm / min at 37°C for 40 hours. Collect the thalli and use MgSO with a concentration of 0.6mol / L 4 The solution was washed 1-2 times, and finally resuspended with 5-10ml OSMO.

[0028] 2. Protoplast preparation: take 100-120mg of Lysing Enzymes from Trichodermaharzianum to...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for improving the gene knockdown conversion rate of talaromyces marneffei, and relates to the field of biotechnology. The method comprises the steps of (1) talaromycesmarneffei strain preparation; (2) protoplast preparation, wherein a novel wall breaking enzyme is mixed with the strain, and cell walls of the strain are dissolved and digested for 1.5-2h; (3) protoplast transformation; (4) protoplast rejuvenation, wherein transformed protoplasts are resuspended in a STC buffer, an SD culture medium containing 1-1.5 mol/L of sorbitol is added, shaking culture isconducted at 23-28 DEG C in a shaker at 80-120 rpm/min for 1-2 days for growth resuming, then spore suspension is inoculated into an ANM-U solid medium, and positive transformants are screened out tocomplete cloning screening. Through the method, the gene conversion efficiency can be improved, and a solid guarantee for researches on gene functions of the talaromyces marneffei is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel gene knockout method for T. marneffei. Background technique [0002] T. marneffei is prevalent in southern my country and Southeast Asia. It is one of the most common opportunistic infections among AIDS patients in endemic areas. It is reported that 19% of AIDS patients in Guangxi are infected with T. marneffei, and the mortality rate is as high as 80%, is an important factor leading to the death of local AIDS patients. In recent years, due to the widespread use of immunosuppressants, T. marneffei infection has become more frequent in patients without overt immunodeficiency. However, little is known about the pathogenic mechanism of T. marneffei at home and abroad. [0003] Gene knockout technology is the basis for studying the infection mechanism, immune escape mechanism and gene function of T. marneffei. The cell wall of T. marneffei is hard to digest; The medium is easil...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N1/15C12R1/80
CPCC12N1/14C12N1/145C12R2001/80
Inventor 曹存巍廖万清郑艳青罗宏潘开素
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGXI MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products