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

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

Method for Intracellular Expression of Human Apolipoprotein ApoAI in Pichia pastoris

technical field

The invention belongs to the technical field of bioengineering, in particular to a method for expressing human apolipoprotein ApoA I. Specifically, by constructing a recombinant engineering strain of Pichia pastoris, the rApoA I protein is highly expressed in yeast cells through shake flask fermentation and induction.

Background technique

Human apolipoprotein ApoA I (apolipoprotein AI) is composed of 243 amino acid residues and has a molecular weight of 28.3KD. It is mainly synthesized in the liver and is an important component of high-density lipoprotein (HDL) and also the function of HDL. the main executor. ApoA I plays an important role in the transport and metabolism of cholesterol, anti-atherosclerosis, anti-endotoxin, anti-inflammation, anti-virus and inhibition of tissue damage caused by acute phase inflammatory response, so it has become one of the focuses of lipid metabolism research one.

Current studies have shown that there are high-affinity ApoA I and HDL receptors on the cell membranes of liver and surrounding tissue cells, and it was found that the liver selectively uptakes HDL cholesteryl esters. Therefore, free HDL and ApoA I have the characteristics of liver targeting, which makes ApoA I have a good application prospect in the research of liver-targeting drugs.

HDL and ApoA I are mainly isolated and extracted from human serum, but the yield is low, the cost is high, and due to the shortage of human blood supply and the difficulty in solving blood pollution, it is difficult to achieve large-scale production, which greatly limits its development and application. Construction of recombinant ApoA I engineering strains with genetic engineering methods is the best means for large-scale production of ApoA I proteins. At present, some people abroad express rApoA I with Escherichia coli, but the expression level is low, about 20mg/L. Some people in China express rApoA I with the insect baculovirus expression system. Although the expression level is relatively high, it cannot be produced on a large scale, and the cost of the culture medium is also very high. Since the Pichia expression system has the characteristics of eukaryotic post-translational processing, and the fermentation conditions are simple, the expression level is high, and the activity of the expression product is good, therefore, the present invention selects the Pichia expression system to carry out the human apolipoprotein gene apoA The expression research of I, as a result, the research obtains important breakthrough, wherein the expression level of secretory expression bacterial strain reaches about 180mg/L (shake flask), and the expression level of intracellular expression bacterial strain reaches about 40mg/L (shake flask), from From the perspective of maintaining the integrity of the ApoA I protein molecule, intracellular expression is better than secretory expression. At present, there are no reports on the expression of rApoAI by Pichia pastoris at home and abroad.

Contents of the invention

The object of the present invention is to provide a method for expressing human apolipoprotein ApoA I with high expression level and low production cost.

The expression method of human apolipoprotein ApoA I proposed by the present invention is that the artificially synthesized apoA I gene is inserted into the intracellular expression plasmid pPIC3.5k (product of INVJTROGEN company); after linearization, electric shock imports Pichia yeast GS115 (product of INVJTROGEN company) In , the recombinant engineered strain was obtained; the intracellular expression of rApoAI was realized through shake flask fermentation and methanol induction. Yeast cells were collected and detected by SDS-PAGE and Western Blotting, which proved that there was obvious expression of rApoA I protein in yeast cells, and its molecular mass was the same as that of ApoA I extracted from human plasma.

Concrete operation steps of the present invention are as follows:

(1) Construction of recombinant expression plasmids

The artificially synthesized apoA I gene was inserted into the pPIC9k plasmid (product of INVJTROGEN Company) to obtain the pPIC9k-apoAI recombinant plasmid, which was used as a template to design primers for PCR amplification. The PCR product containing the apoA I gene was double-digested with EcoRI and BamH I, and the intracellular expression plasmid pPIC3.5k was also double-digested with EcoR I and BamH I, and then ligated with T4 DNA ligase to obtain the recombinant expression plasmid pPIC3.5k- apoA I;

(2) Screening recombinant engineering strains

The recombinant expression plasmid pPIC3.5k-apoA I was cut with Bgl II, transformed into Pichia pastoris GS115 by electric shock, the transformation solution was spread on the RDB plate, and the transformant was obtained after culturing; the transformant was screened by G418 resistance to obtain a highly resistant strain ; The G418 highly resistant strain was identified by PCR, and the positive clone was the recombinant engineering strain GS115/pPIC3.5k-apoA I;

(3) Shake flask fermentation

The highly expressed recombinant engineered strains were inoculated in BMGY medium for growth, and then transferred to BMMY medium for methanol induction to express rApoA I in the cells.

(4) SDS-PAGE detection of rApoA I

Get the centrifugation of fermented liquid, collect thalline, detect with 15% SDS-PAGE, have the expression of obvious rApoA I protein, (expression level is about 40mg/L).

(5) Western blotting detection of rApoA I

The primary antibody in Western blotting is Rabbit anti-Human ApolipoproteinA-I, and the secondary antibody is Goat anti-Rabbit IgG (Alkaline phosphate conjugated).

In view of the fact that human apolipoprotein ApoA I has anti-atherosclerosis, anti-endotoxin and anti-inflammation functions, and has a good application prospect in the study of liver-targeted drugs, the successful expression of rApoA I by Pichia pastoris is a good way for future research. The development of ApoA I into a class of new drugs or as a carrier for targeted drugs has laid the foundation.

Description of drawings

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

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

The SDS-PAGE electrophoresis collection of Fig. 3 the present invention: m, protein molecular mass standard; 1.GS115 cell extract; 2,3,4, pPIC3.5k-apoA I cell extract; 5, extract in the human blood plasma ApoA I; 6, GS115/pPIC9k-apoAI fermentation supernatant.

Figure 4 Western blotting detection of the present invention: 1, GS115/pPIC9k-apoA I fermentation supernatant; 2, GS115/pPIC3.5k-apoA I cell extract; 3, ApoA I extracted in human plasma; 4, GS115 cell extract.

Detailed ways

Embodiment 1, Construction and Identification of Recombinant Expression Plasmids

Using the plasmid pPIC9k-apoA I with the artificially synthesized gene apoA I as a template, primers were designed for PCR amplification. Forward primer: 5'-GGGGATCCAAACGATGGATGAACCACCTCAGTCTCCATGG-3', BamH I site was introduced at the 5' end, reverse primer: 5'-GCAAATGGCATTCTGACATCC-3'. The PCR reaction conditions were set according to conventional settings. The PCR product was double-digested with EcoR I and BamH I, and the intracellular expression plasmid pPIC3.5k was also double-digested with EcoR I and BamH I, and then ligated and transformed. The recombinant expression plasmid pPIC3.5k-apoA I was extracted from the transformant, identified by enzyme digestion and DNA sequence determination, which proved that the construction of the recombinant expression plasmid was completely correct.

Example 2, Electrotransformation of recombinant expression plasmid Pichia GS115

Inoculate Pichia yeast GS115(his4) in YPD medium, shake culture at 28-30°C until OD ₆₀₀ =1.3-1.5, centrifuge, collect the bacteria, wash once with pre-cooled sterile water and 1mol/L sorbitol , and finally suspended in 200 μl of 1mol/L sorbitol to obtain GS115 competent cells. Take 80 μl GS115 competent cells and mix 5 μg recombinant expression plasmid digested with Bg1 II, transform by electroporation at 1300V, 25μF, 200Ω, suspend with 1ml of 1mol/L sorbitol, spread on RDB plate, and culture at 28-30℃ After 3-4 days, more than 1400 transformants were obtained as a result.

Example 3, Screening of G418 highly resistant yeast transformants

More than 1,400 colonies of yeast transformants grown on RDB plates were planted on YPD plates with G418 concentrations of 1.5, 3.0, and 4.0 mg/ml, cultured at 28-30°C, and G418-resistant strains were screened step by step. Results 27 strains were obtained on the plate containing 4mg/ml G418.

Embodiment 4, the PCR identification of yeast transformant

The colony of the above-mentioned G418 highly resistant strain was taken and placed in sterile water, mixed evenly and boiled, centrifuged and the supernatant was taken for PCR. See Example 1 for primers. The PCR reaction conditions were set according to conventional settings. The positive clone is the recombinant engineering strain GS115/pPIC3.5k-apoA I

Embodiment 5, the screening of highly expressed recombinant engineered bacteria

Inoculate the recombinant engineered strain GS115/pPIC3.5k-apoA I into a test tube containing 3ml of BMGY, culture with shaking at 28-30°C for 16-20 hours, take 1.5ml of bacterial liquid and transfer it to 50ml of BMGY medium (shake flask), 28-30 Cultivate with shaking at ℃ for 22-26 hours, centrifuge the bacterial solution, and collect the bacterial cells. Use 15ml BMMY medium (shake flask) to suspend the bacteria, shake and culture at 28-30°C for 70-75 hours, and add methanol once every 24 hours. The fermentation broth was centrifuged to collect the bacteria, and detected by 15% SDS-PAGE. As a result, 4 high-expressing strains were obtained, and No. 5 strain was selected as the experimental strain. The rApoA I expressed in the cell had the same molecular mass as the ApoA I isolated from human plasma, and rApoA I accounted for 7.86% of the total soluble protein of the bacteria. The expression level is about 40mg/L.

Embodiment 6, the Western blotting identification of expression product

The electrophoresis SDS-PAGE gel was stripped, electrotransferred to the NC membrane, and the NC membrane was blocked with 5% skimmed milk; the primary antibody (Rabbit anti-Human ApolipoproteinA-I) was added, shaken at 37°C for 1 hour, and washed twice; Anti-(Goatanti-Rabbit IgG (Alkaline phosphatase conjugated), shaking at 37°C for 1h, washing once; washing once with substrate buffer; adding 33μl BCIP and 66μl NBT to 10ml alkaline phosphatase substrate buffer, the NC Immerse the membrane and develop color at 37°C for 3 minutes; rinse with water to terminate the reaction.

Claims (1)

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 '.

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