CN101921798A

CN101921798A - Method for preparing gene recombinant human ferritin light chain

Info

Publication number: CN101921798A
Application number: CN2009101419740A
Authority: CN
Inventors: 张曼
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-06-12
Filing date: 2009-06-12
Publication date: 2010-12-22

Abstract

The invention discloses a method for preparing a gene recombinant human ferritin light chain. The method comprises the following steps of: amplifying a coding genetic sequence of a ferritin light chain (Ferritin L) in human fresh complete blood cells by PCR, cloning the amplified coding genetic sequence into an expression vector pET-30a by enzyme digestion and connection, transforming E.coli BL21 (DE3), identifying a positive cloning colony, performing induction expression on the Ferritin L by IPTG, and analyzing the antigenicity of the Ferritin L by using Westernblot. The method successfully constructs the prokaryotic expression vector of the FTL, and obtains the pure ferritin light chain by induction expression and purification in colon bacillus. Proved by the Westernblot, the protein has good antigenicity.

Description

A kind of preparation method of gene recombinant human ferritin light chain

Technical field

The invention belongs to technical field of bioengineering, be specifically related to a kind of preparation method of gene recombinant human ferritin light chain.

Background technology

Clinical medical development is more and more higher to accuracy, stability and the susceptibility requirement of assay, and therefore high-quality quality control product, calibration object and reference material are the critical products in the check diagnosis.China is the ferritin product that the application first-generation and the s-generation are organized purifying substantially at present, and its activity, linearity, stability, specificity, susceptibility, shelf time etc. are all widely different owing to the uncertainty in source.Therefore, gene recombination method is used rapidly and is promoted this series products and enters the third generation.Because the ferritin complex structure, the common renaturation of 24 subunit units is extremely difficult, obtain that minimum detectable level, height are tired, the linear gene recombination ferritin standard substance that require is technical very high on a large scale.Therefore, China does not still have gene recombination ferritin standard substance at present.

Because ferritin and inflammatory disorders, hemopathy, hepatopathy, cardiovascular and cerebrovascular diseases, ephrosis and malignant tumour etc. are closely related, in the clinical diagnosis of these diseases, treatment, monitoring, all play important effect.Therefore, study to guaranteeing restriction, stable in properties, the differences between batches gene recombination ferritin standard substance with China's independent intellectual property right little, that use cost is lower that the quality inspection research and development are not originated extremely urgent.Particularly the ferritin detection method of using clinically at present is varied, and the consistence between the whole bag of tricks is difficult to comparison.Domestic expert appeals to make up the ferritin reference material of China oneself already, in the hope of real accuracy and the comparability that solves ferritin mensuration.This research and design a kind of with the method for ferritin light chain (Ferritin L) at expression in escherichia coli, identify through order-checking and Westernblot, confirm to have good antigenicity.Now the result is reported as follows.

Summary of the invention

A technical problem to be solved by this invention provides a kind of preparation method of gene recombinant human ferritin light chain.For production purity ferritin reference material good, stable, that easily store and transport is set up the basis.

For solving the problems of the technologies described above, technical scheme provided by the present invention is:

The invention provides a kind of preparation method of gene recombinant human ferritin light chain, it is characterized in that may further comprise the steps:

(1) adopt the RT-PCR technology from the human blood cell, to obtain the human ferritin light chain gene fragment;

(2) the human ferritin light chain gene fragment subclone that PCR in the step (1) is obtained is to the pGEM-T carrier, transformed competence colibacillus Top10 bacterium, be inoculated in the LB substratum that contains penbritin then, the screening positive clone bacterial strain, extract the plasmid pGEM-T/ferritin L of positive colony bacterial strain, and the evaluation of checking order.

(3) be template with the plasmid pGEM-T/ferritin L in the step (2), carry out pcr amplification, then amplified production is carried out double digestion, simultaneously empty plasmid pET-30a is carried out double digestion, recovery and purifying enzyme are cut product respectively, connect with the T4 ligase enzyme, obtain expression vector, the transformed competence colibacillus intestinal bacteria, be inoculated in the LB substratum that contains kantlex then, the screening positive clone bacterial strain, the expression plasmid pET-30a/ferritin L of extraction positive colony bacterial strain, and the evaluation of checking order.

(4) express the purifying ferritin light chain with the IPTG inducible protein.

Wherein, intestinal bacteria are BL21 or DE3 described in the step (3).

Double digestion is for to carry out double digestion with NdeI and BamHI described in the step (3).

The advantage that the present invention has: at present, China's ferritin national standard product still obtain by purifying.The development of ferritin reference material can solve the disadvantage that the present ferritin reference material of China relies on import for a long time, can set up the reference material of China, and is that the system of tracing to the source of setting up and improve the ferritin detection lays the first stone; For the accuracy, consistence and the reliability that will improve clinical ferritin detection are from now on given security, play important effect for improving China's ferritin observed value comparability, equivalence in the world simultaneously.The present invention is the method for utilization gene recombination, give expression to the light chain of ferritin respectively, and resulting albumen is carried out initial analysis identify, determine the feasibility of ferritin gene reorganization reference material research and development, and provide new thinking for the research and development of clinical other reference material.

Description of drawings

Fig. 1 pET-30a/ferritin L gene amplification product electrophorogram.(1DNA marker; 2pET-30a/ferritin L PCR product)

Fig. 2 pET-30a/ferritin L SDS-PAGE electrophorogram.(1 albumen marker; 2pET-30a/ferritin L expression product)

Embodiment

Adopt embodiment that the present invention is described in further detail below.

Bacterial classification and plasmid

Competence intestinal bacteria Top10 is available from Tiangen company; PGEM-T cloning vector test kit is available from match Parkson, Beijing company; E. coli bl21 (DE3) bacterial strain and expression vector pET-30a are available from Novagen company.

Main agents

Taq archaeal dna polymerase and primer are available from match Parkson, Beijing company; Plasmid extracts test kit, total RNA extraction reagent box and RT-PCR test kit in a small amount available from Beijing Tiangen company; Restriction enzyme is available from NEB company; With toolenzyme such as T4 ligase enzyme available from match Parkson, Beijing company; DNA reclaims test kit available from Tiangen company; Mouse-anti people Ferritin L monoclonal antibody is available from Santa cruz company; The sheep anti-mouse igg of HRP mark is available from the match biology of speeding; Other reagent is homemade or the import analytical pure.

The preparation of embodiment 1 subclone plasmid pGEM-T/ferritin L

Extract total RNA with the Trisol test kit from people's fresh whole blood cell, adopt the RT-PCR technology to obtain the cDNA fragment of human ferritin light chain gene (Ferritin L) from people's fresh whole blood cell, according to the operation of test kit specification sheets, the primer is:

Upstream primer: 5 '-CATATGAGCTCCCAGATTCGTCAG-3 ';

Downstream primer: 5 '-GGATCCTCTTAGTCGTGCTTGAGAG-3 '

Reclaim and purified pcr product after, be connected in 4 ℃ with the pGEM-T carrier and spend the night, transformed competence colibacillus Top10 bacterium inoculates the LB culture dish that contains penbritin, screening positive clone bacterial strain, and the evaluation of checking order.This bacterial strain is the intestinal bacteria that contain Ferritin L encoding sequence plasmid pGEM-T/ferritin L.

The structure of embodiment 2 expression vector pET-30a/ferritin L

With plasmid pGEM-T/FTL is template, carries out double digestion with NdeI and BamHI; Simultaneously with empty plasmid pET-30a NdeI+BamHI double digestion.Reclaim above-mentioned enzyme respectively and cut product, after mixing with suitable proportion, spend the night in 16 ℃ of connections with the T4 ligase enzyme, transformed competence colibacillus BL21 (DE3) bacterium, inoculation contains the LB culture dish of kantlex, screening positive clone bacterial strain.

The plasmid pET-30a/ferritin L that extracts the positive colony bacterial strain evaluation of checking order.Inserting codified among segmental dna sequence dna and the GenBank (among the GenBank number), to contain 175 amino-acid residue ferritin L sequences in full accord, and single open reading frame is correct, the required target protein of codified.As shown in Figure 1.

Embodiment 3Ferritin L induction expression of protein

The engineering bacteria BL21 (DE3) that will contain pET-30a/ferritin L plasmid is inoculated in 5mI and contains in the LB substratum of kantlex (50ml), incubated overnight, press 1: 50 transferred species next day to 1L LB substratum (kalamycin resistance), 37 ℃, about 240r/min rotating and culturing 2h, make A600 reach 0.6-0.8, add inductor IPTG, continue jolting 4h under these conditions to final concentration 1mmol/L.After cultivating end, the centrifugal 10min of 8000g collects bacterial sediment.With PBS washing 3 times, ice-bath ultrasonic cracking thalline, the centrifugal 20min of 12000g, cleer and peaceful precipitation in the separation.

The expressing protein of pET-30a/ferritin L is analyzed through SDS-PAGE, and as shown in Figure 2, visible relative molecular mass is about 20000 expressing protein, and is consistent with the expection size.Handle the back discovery through ultrasonic degradation, target protein is mainly with the formal representation of soluble proteins.

The proteic Westernblot of embodiment 4Ferritin L identifies

Through 10%SDS-PAGE, electricity is transferred on the nitrocellulose filter with Ferritin L, uses mouse-anti people Ferritin L and Ferritin H monoclonal antibody (one is anti-) and HRP-sheep anti-mouse igg (two is anti-) to detect proteic two kinds of antigenicities respectively, develops the color with DAB at last.Ferritin L albumen and two kinds of reaction systems of Ferritin L monoclonal antibody+HRP one sheep anti-mouse igg of expressing, the visible obviously colour developing band at relative molecular mass 20000 places.Show that target protein has good antigenicity.

Claims

1. the preparation method of a gene recombinant human ferritin light chain is characterized in that may further comprise the steps:

(2) the human ferritin light chain gene fragment subclone that PCR in the step (1) is obtained is to the pGEM-T carrier, transformed competence colibacillus Top10 bacterium, be inoculated in the LB substratum that contains penbritin then, the screening positive clone bacterial strain, extract the plasmid pGEM-T/ferritin L of positive colony bacterial strain, and the evaluation of checking order;

(3) be template with the plasmid pGEM-T/FTL in the step (2), carry out pcr amplification, then amplified production is carried out double digestion, simultaneously empty plasmid pET-30a is carried out double digestion, recovery and purifying enzyme are cut product respectively, connect with the T4 ligase enzyme, obtain expression vector, the transformed competence colibacillus intestinal bacteria, be inoculated in the LB substratum that contains kantlex then, the screening positive clone bacterial strain, the expression plasmid pET-30a/ferritin L of extraction positive colony bacterial strain, and the evaluation of checking order;

(4) express the purifying ferritin light chain with the IPTG inducible protein.

2. the preparation method of gene recombinant human ferritin light chain according to claim 1, it is characterized in that: intestinal bacteria are BL21 or DE3 described in the step (3).

3. the preparation method of gene recombinant human ferritin light chain according to claim 1, it is characterized in that: double digestion is for to carry out double digestion with NdeI and BamHI described in the step (3).