CN1320119C

CN1320119C - Method of expressing human apolipoprotein ApoA I inside Pichia yeast cell

Info

Publication number: CN1320119C
Application number: CNB2004100893154A
Authority: CN
Inventors: 宋大新; 赵志安; 杨婷婷; 张淼; 张彦
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2004-12-09
Filing date: 2004-12-09
Publication date: 2007-06-06
Anticipated expiration: 2024-12-09
Also published as: CN1637145A

Abstract

The present invention belongs to the technical field of biological engineering, more specifically a method of expressing human apolipoprotein ApoA I inside Pichia yeast cells. ApoA I genes artificially synthesized are inserted into expression plasmids pPIC3.5k in cells; Pichia yeast strains GS115 are led in by electroshock; obvious rApoA I protein expression exists in yeast cells by the fermentation of a shake flask and carbinol induction; molecular mass is identical with that of ApoA I extracted in human plasma.

Description

The method that human apolipoprotein ApoA I is expressed in the pichia born of the same parents

Technical field

The invention belongs to technical field of bioengineering, be specifically related to the expression method of a kind of human apolipoprotein ApoA I.Specifically be by making up the pichia recombinant strain, in yeast cell, efficiently expressing rApoA I albumen with inducing through shake flask fermentation.

Background technology

Human apolipoprotein ApoA I (apolipoproteinAI) is made up of 243 amino-acid residues, molecular mass is 28.3KD, and is mainly synthetic in liver, is high-density lipoprotein (HDL) (high-density lipoprotein, HDL) important component also is the main executive of HDL function.ApoA I is at the transhipment of cholesterol and metabolism, atherosclerosis, antiendotoxin, anti-inflammatory, antiviral and suppress to play an important role in the tissue injury that acute inflammatory reaction mutually causes, so it has become one of emphasis of lipid metabolism research.

Present studies show that, has the ApoA I and the HDL acceptor of high-affinity on the cytolemma of liver and surrounding tissue cell, and finds liver selectivity picked-up HDL cholesteryl ester.Therefore free HDL and ApoA I have the characteristics of liver target, and these characteristics make ApoA I in the liver target drug research good prospects for application be arranged.

HDL and ApoA I be separation and Extraction from human serum mainly, but yields poorly, the cost height, and since human blood short supply and be difficult to solve problem such as blood contamination be difficult to realize scale operation, so limited its development and application greatly.Making up reorganization ApoA I engineering strain with gene engineering method, is the proteic best means of scale operation ApoA I.Human escherichia coli expression rApoA I is arranged at present, but expression level is low, is about 20mg/L abroad.Domestic have the human insect baculovirus expression system to express rApoA I, though expression level is higher, can't scale operation, and the nutrient solution cost is also very high.Because the pichia expression system has the characteristics of the translation post-treatment of eukaryotic system, and fermentation condition is simple, the expression level height, the expression product activity is good, therefore, the present invention selects for use the pichia expression system to carry out the expression study of human apolipoprotein gene apoA I, the result, research obtains important breakthrough, wherein the expression level of secretion type expression bacterial strain reaches about 180mg/L and (shakes bottle), and the expression level of expression strain reaches and (shakes bottle) about 40mg/L in the born of the same parents, from keeping the integrity angle of ApoA I protein molecular, expresses in the born of the same parents and is better than secreting, expressing.There is no both at home and abroad at present and adopt pichia to express the report of rApoAI.

Summary of the invention

The expression method that the purpose of this invention is to provide a kind of expression level height, human apolipoprotein ApoA I that production cost is low.

The expression method of the human apolipoprotein ApoA I that the present invention proposes is with expression plasmid pPIC3.5k (INVJTROGEN company product) in the apoA I gene insertion born of the same parents of synthetic; Electric shock imports and finishes among the redization yeast GS115 (INVJTROGEN company product) after the linearizing, obtains recombinant strain; Through shake flask fermentation and methanol induction, realize interior expression of born of the same parents of rApoAI.Collect yeast cell, detect with SDS-PAGE and Western Blotting, proving has tangible rApoA I protein expression in the yeast cell, and the ApoA I that extracts in its molecular mass and the human plasma is identical.

Concrete operations step of the present invention is as follows:

(1) makes up recombinant expression plasmid

The apoA I gene of synthetic is inserted pPIC9k plasmid (INVJTROGEN company product), obtain the pPIC9k-apoAI recombinant plasmid, as template, the design primer carries out pcr amplification.To contain PCR the product EcoRI and the BamH I double digestion of apoA I gene, expression plasmid pPIC3.5k also uses EcoR I and BamH I double digestion in the born of the same parents, connects with the T4DNA ligase enzyme then, obtains recombinant expression plasmid pPIC3.5k-apoA I;

(2) screening recombinant strain

Recombinant expression plasmid pPIC3.5k-apoA I is cut through Bgl II, transform pichia GS115 with electric shock, conversion fluid coating RDB flat board is cultivated the back and is obtained transformant; Transformant obtains high resistant strain through the G418 resistance screening; G418 is high, and resistant strain is identified through PCR, and positive colony is recombinant strain GS115/pPIC3.5k-apoA I;

(3) shake flask fermentation

The recombinant strain of high expression level is inoculated in the growth of BMGY substratum, changes over to then and carry out methanol induction in the BMMY substratum, make and express rApoA I in the born of the same parents.

(4) SDS-PAGE of rApoA I detects

It is centrifugal to get fermented liquid, collects thalline, detects with 15%SDS-PAGE, the proteic expression of tangible r ApoA I is arranged, (expression level is about 40mg/L).

(5) the Western blotting of rApoA I detects

Among the Western blotting one be anti-to be Rabbit anti-Human ApolipoproteinA-I, and two anti-ly are Goat anti-Rabbit IgG (Alkaline phosphatase conjugated).

In view of human apolipoprotein ApoA I has functions such as atherosclerosis, antiendotoxin and anti-inflammatory, and in the liver target drug research, good prospects for application is arranged, therefore adopt pichia successful expression rApoA I, for from now on ApoA I being developed to a kind new medicine or, laying a good foundation as the carrier of targeted drug.

Description of drawings

The design of graphics of Fig. 1 recombinant expression plasmid pPIC3.5k-apoA of the present invention I

The PCR of Fig. 2 yeast transformant of the present invention identifies: 1.DL-2000 Marker; 2,3, pPIC3.5k-apoAI; 4, host bacterium GS115; 5, GS115/pPIC9k-apoA I.

Fig. 3 SDS-PAGE electrophoretogram of the present invention: m, protein molecular quality standard; 1.GS115 cell extract; 2,3,4, pPIC3.5k-apoA I cell extract; 5, the ApoA I that extracts in the human plasma; 6, GS115/pPIC9k-apoAI fermented supernatant fluid.

Fig. 4 Western blotting of the present invention detects: 1, GS115/pPIC9k-apoA I fermented supernatant fluid; 2, GS115/pPIC3.5k-apoA I cell extract; 3, the ApoA I that extracts in the human plasma; 4, GS115 cell extract.

Embodiment

The structure of embodiment 1, recombinant expression plasmid and evaluation

With the plasmid pPIC9k-apoA I that has synthetic gene apoA I is template, and the design primer carries out pcr amplification.Forward primer: 5 '-GGGGATCCAAACGATGGATGAACCACCTCAGTCTCCATGG-3 ', 5 ' end is introduced BamH I site, reverse primer: 5 '-GCAAATGGCATTCTGACATCC-3 '.The PCR reaction conditions is provided with routinely and carries out, and the PCR product is with EcoR I and BamH I double digestion, and expression plasmid pPIC3.5k also uses EcoR I and BamH I double digestion in the born of the same parents, connects then and transforms.Extract recombinant expression plasmid pPIC3.5k-apoA I from transformant, enzyme is cut and is identified and determined dna sequence, proves that the structure of recombinant expression plasmid is entirely true.

Embodiment 2, recombinant expression plasmid electricity transform pichia GS115

Inoculation pichia GS115 (his4) is in the YPD substratum, and 28-30 ℃ of shaking culture is to 0D ₆₀₀=1.3-1.5, centrifugal, collect thalline, respectively wash once with precooling sterilized water and 1mol/L sorbyl alcohol, suspend with 1mol/L sorbyl alcohol 200 μ l at last, promptly obtain the GS115 competent cell.Get the recombinant expression plasmid mixing that 80 μ l GS115 competent cells and 5 μ g cut with Bg1 II enzyme, at 1300V, 25 μ F; electric shock transforms under the 200 Ω conditions, after suspending with 1mol/L sorbyl alcohol 1ml, and coating RDB flat board; cultivated 3-4 days for 28-30 ℃, the result obtains more than 1400 transformant.

The screening of the high resistance yeast transformant of embodiment 3, G418

Will G418 concentration be 1.5,3.0 to containing in more than 1400 the yeast conversion daughter colony dibbling of growing on the RDB flat board, on the YPD flat board of 4.0mg/ml, 28-30 ℃ of cultivation, step-sizing G418 resistant strain.The result is containing 27 bacterial strains of acquisition on the 4mg/mlG418 flat board.

The PCR of embodiment 4, yeast transformant identifies

The bacterium colony of getting the high resistant strain of above-mentioned G418 places sterilized water, and mixing and boiling is got supernatant liquor after centrifugal and carried out PCR.Primer is referring to embodiment 1.The PCR reaction conditions is provided with routinely and carries out.Positive colony is recombinant strain GS115/pPIC3.5k-apoA I

The screening of embodiment 5, high expression level recombinant bacterial strain

Recombinant strain GS115/pPIC3.5k-apoA I is inoculated in the test tube that contains 3mlBMGY, 28-30 ℃ shaking culture 16-20 hour, get 1.5ml bacterium liquid and change in the 50ml BMGY substratum (shake bottle), 28-30 ℃ shaking culture 22-26 hour, bacterium liquid is centrifugal, collects thalline.With 15ml BMMY substratum (shake bottle) suspension thalline, 28-30 ℃ shaking culture 70-75 hour, added one time methyl alcohol in per 24 hours.It is centrifugal to get fermented liquid, collects thalline, detects with 15%SDS-PAGE.The result obtains 4 plant height expression strains, and wherein No. 5 bacterial strains are as experimental strain in choosing, and the molecular mass of the rApoA I that expresses in its born of the same parents is identical with isolating ApoA I in the human plasma, and r ApoA I accounts for 7.86% of thalline total soluble protein, and expression level is about 40mg/L.

The Western blotting of embodiment 6, expression product identifies

Electrophoretic SDS-PAGE gel is peeled off, and electrotransfer is to the NC film, and the NC film seals with 5% skimmed milk; Add one anti-(Rabbit anti-Human ApolipoproteinA-I), 37 ℃ of vibration 1h wash 2 times; Add two anti-(Goatanti-Rabbit IgG (Alkaline phosphatase conjugated), 37 ℃ of vibration 1h wash 1 time; With substrate buffer solution washing 1 time; In 10ml alkaline phosphatase substrate damping fluid, add 33 μ l BCIP and 66 μ l NBT, the NC film is immersed, 37 ℃ of colour developing 3min; Water flushing termination reaction.

Claims

1, the method expressed in the pichia born of the same parents of a kind of human apolipoprotein ApoA I is characterized in that the apoAI gene of synthetic is inserted expression vector pPIC3.5k in the born of the same parents; Electric shock imports among the pichia GS115 after the linearizing, obtains recombinant strain; Through shake flask fermentation and methanol induction, realize interior expression of born of the same parents of human apolipoprotein ApoA I; The concrete operations step is as follows:

(1) makes up recombinant expression plasmid

The gene apoA I of synthetic is inserted plasmid pPIC9k, obtain recombinant plasmid pPIC9k-apoA I, as template, the design primer carries out pcr amplification, PCR product the EcoR I and the BamH I double digestion that will contain apoA I gene, expression plasmid pPIC3.5k also uses EcoR I and BamH I double digestion in the born of the same parents, connects with the T4DNA ligase enzyme then, obtains recombinant expression plasmid pPIC3.5k-apoA I;

(2) screening recombinant strain

Recombinant expression plasmid pPIC3.5k-apoA I is cut through Bgl II enzyme, transform pichia GS115 with electric shock, conversion fluid coating RDB flat board is cultivated the back and is obtained transformant; Transformant obtains high resistant strain through the G418 resistance screening; G418 is high, and resistant strain is identified through PCR, and positive colony is recombinant strain GS115/pPIC3.5k-apoA I;

(3) shake flask fermentation

The recombinant strain of high expression level is inoculated in the growth of BMGY substratum, changes over to then and carry out methanol induction in the BMMY substratum, make expressing human aPoA poA I in the born of the same parents;

Wherein, the PCR primer sequence of design is as follows:

Forward primer: 5 '-GGGGATCCAAACGATGGATGAACCACCTCAGTCTCCATGG-3 '

Reverse primer: 5 '-GCAAATGGCATTCTGACATCC-3 '.