Preparation method of secretory type Rpf (Resuscitation promoting factor) factor based on transformant and application thereof

Abstract

The invention discloses a preparation method of a secretory type Rpf factor based on a transformant and application thereof. On the basis of constructing an exogenous expression vector pPIC-Rpf, the exogenous expression vector is converted into Pichia methanolica to prepare a recombinant, and the Pichia methanolica recombinant of a secretory type Rpf protein is obtained by resistance screening. APichia methanolica expression system can persistently secrete the Rpf protein with biologic activity; the protein can promote the resuscitation of mycobacterium smegmatis dormant bacteria and the growth of Mycobacterium tuberculosis (MTB) H37Ra, promotes the growth of the MTB in the sputum specimens of clinical TB (tuberculosis) patients and can be used for the rapid diagnosis of clinical TB.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and relates to the expression and purification of a bacterial revival-promoting factor (Rpf factor), in particular to a preparation method and application of a transformant-based secreted Rpf protein. Background technique [0002] 1. Function and structure of Rpf factor [0003] Rpf (Resuscitation-promoting factor, Rpf) is a kind of bacterial resurrection promoting factor, its function is similar to the growth factor of eukaryotes, can be in pg (10 -12 ) levels promote the resurrection and growth of dormant bacteria in autocrine and paracrine forms, so they are called bacterial growth factors. This factor was originally discovered in Micrococcus luteus (M.luteus). In addition to promoting the resurrection and growth of dormant bacteria, Rpf factor also has a corresponding effect on normal growing bacteria; the mechanism may be involved in the signal transduction between cells guide...

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Problems solved by technology

The detection rate of isolation culture is also very low, especially for persistent bacteria, dormant bacteria and metabolic heterogeneous bacteria that cannot be successfully cultured

[0009] In recent years, several new diagnostic methods have been tried at home and abroad, such as detecting anti-MTB antibodies or detecting MTB nucleic acid by PCR to assist in the diagnosis of tuberculosis. The sensitivity of these methods is higher than that of the aforementioned methods, but the detection rate is generally lower. Only around 70%, and the false positive rate is also high

At the same time, due to the emergence of a large number of drug-resistant strains, the treatment needs the guidance of drug-sensitive experiments. Although methods such as gene chips and single-strand conformation polymorphism can be used to quickly identify drug-resistant strains, some genetic mutations that cause drug resistance are still unknown. Unclear, some mutation sites are nonsense mutations, new antibiotics are used, etc., making the sensitivity less than 80%, and the test results must be verified by phenotypic methods

The main problems of conventional MTB isolation and culture in clinical testing are low sensitivity and long time required

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