CN102020712B - Human-like collagen for vaccine stabilizing agent and production method thereof - Google Patents

Abstract

The invention relates to a human-like collagen for a vaccine stabilizing agent and a production method thereof, and belongs to the technical field of the vaccine stabilizing agents. The human-like collagen is obtained by fermenting yeast engineering bacteria which are named pichia pastoris X-33/col CGMCC NO.4187. The production method comprises the following steps of: artificially synthesizing human-like collagen genes; building and screening the yeast engineering bacteria containing the human-like collagen genes; inducing and culturing the engineering bacteria by shaking a flask; and detecting the target human-like collagen by using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The human-like collagen has the advantages that the human-like collagen produced by utilizing the yeast genetic engineering bacteria not only has favorable biological characteristics, but also has the good properties of no virus hidden danger, low rejection reaction and the like compared with the product derived from animals, so the purified human-like collagen can be applied to the vaccine stabilizing agent in stead of the collagen derived from the animals.

Description

A kind of Human-like Collagen and production method thereof that can be used for vaccine stabilizing agent

Technical field

The invention belongs to the vaccine stabilizing agent technical field, particularly relate to a kind of Human-like Collagen and production method thereof that can be used for vaccine stabilizing agent, utilize genetic engineering technique to transform yeast and produce Human-like Collagen.

Background technology

Collagen protein claims again collagen, is the spirality fiber shape protein that is twisted into by three peptide chains, and this proteinoid is the maximum protein of people's in-vivo content.The amino acid of collagen protein forms following feature: 1., glycine (glycine) is about 25～30%, every two other amino-acid residues (X, Y) glycine arranged namely, its peptide chain can with (sweet-X-Y) n represents.2., proline(Pro) (Proline) about 12% in the collagen, oxyproline (hydroxyproline, Hyp) about 10%, hydroxyproline content is few in the general animal protein, L-Ala (alanine) has 11% in addition, hydroxylysine (hydroxylysine) about 0.5%.3., lack tryptophane in the collagen, so it nutritionally is incomplete protein.

Because collagen protein has the characteristics such as nonantigenic, biodegradable, easy absorption, nontoxic and physiologically acceptable, therefore enjoy favor in the medical material field, be widely used in operating sutures, styptic sponge, artificial skin, artificial blood vessel, artificial cornea, pharmaceutical carrier, the aspects such as vaccine stabilizing agent.

A lot of vaccines are very responsive to the condition of preparation and the process of preservation, especially attenuated live vaccine.In order to protect the integrity of biotic component, in the vaccine preparation process, usually add stablizer to guarantee the immunization effect.All the time, can comprise sugar as the material of vaccine stabilizing agent, alkali metal phosphate, glutaminate, ox or human serum albumin, casein hydrolysate etc., topmost in addition gelatin.Gelatin (Gelatin) is the hydrolysate of collagen, is a kind of fat free high protein, does not contain cholesterol, be the stable good selection of protection vaccine, but it mainly extracts acquisition from animal skin.

Because there is viral hidden danger in the collagen protein of animal-origin and easily causes the human body rejection, therefore adopting Protocols in Molecular Biology to prepare recombinant human collagen albumen has the incomparable advantage of various traditional extraction process.At present, the host who utilizes genetic engineering technique to express collagen protein of report has microorganism (intestinal bacteria and yeast), insect, mammalian cell etc. both at home and abroad.Because intestinal bacteria are easily cultivated, breeding is fast, and the example of existing a lot of transgenosis medicine successful expression has now become the carrier that people's preferred genes is expressed.Yet for Human-like Collagen, there is fatal defective in intestinal bacteria, and namely escherichia expression system can't carry out modifying behind the protein translation, so can't form the collagen protein of higher structure.Insect and mammalian cell all are in laboratory level at present, although the recombined collagen that both express on structure function closer to human collagen albumen, the defective such as its cost is high, and the production cycle is long has all hindered again their development.Yeast is eukaryote; the enzyme that possesses molecular chaperone protein and glycosylation, hydroxylation, ethanoyl; can after synthetic, carry out certain modification to some albumen with higher structure; and the culture condition of yeast cell; cost is also much lower than mammalian cell, is fit to very much suitability for industrialized production.

Summary of the invention

The object of the present invention is to provide a kind of Human-like Collagen and production method thereof that can be used for vaccine stabilizing agent, the Yeast engineering bacterium strain that a strain is new, it can utilize the synthetic human-like collagen gene to express the Human-like Collagen of small molecular weight.

This Yeast engineering bacteria called after pichia spp (Pichia sP.), numbering CGMCC No.4187.In China Committee for Culture Collection of Microorganisms of the depositary institution common micro-organisms center preservation of State Intellectual Property Office's appointment, deposit number is CGMCC No.4187 to this bacterial classification, the preservation time: on September 20th, 2010.

For achieving the above object, the technical solution adopted in the present invention is:

1. synthetic human-like collagen gene col, its base sequence is as follows

CCCAGGACCAGCTGGTCAAGATGGACGTCCTGGACCTCCAGGCCCTCCAGGGGCCCGAGGCCAAGCAGGTGTTATGGGGTTTCCTGGACCAAAGGGAGCAGCCGGCGAGCCAGGTAAAGCCGGCGAAAGGGGTGTCCCAGGACCACCAGGTGCTGTGGGACCAGCCGGCAAAGACGGTGAGGCAGGTGCTCAGGGTCCACCAGGACCTGCTGGTCCTGCTGGAGAAAGATAA

Its length nucleic acid is 306bp.

2. the production method of described Human-like Collagen is as follows:

(1) construction process of described Yeast engineering bacteria

1. according to known its gene order of collagen, amino acid sequence synthetic, then carry out the modification of yeast codon preference, finally obtain synthetic human-like collagen gene col.

2. again synthetic human-like collagen gene col is cloned into Yeast expression carrier pPICZ α, called after pPICZ α/col;

3. utilize electric shock transformation method that recombinant plasmid pPICZ alpha/col is converted into yeast cell X-33.

4. utilize at last the correct yeast transformant of method screening of PCR and order-checking.

(2) the shaking flask inducing culture of described Yeast engineering bacteria:

1. the composition of solid medium (YEPD) and every liter of content thereof are: peptone 20 grams, yeast powder 10 grams, glucose 20 grams, agar powder 15 grams.

2. solid culture condition: Yeast engineering bacteria is inoculated in the culture dish of above-mentioned solid medium, cultivated 3-5 days in 20 ℃-50 ℃.

3. the composition of seed culture medium and every liter of content thereof are: peptone 20 grams, yeast powder 10 grams, yeast choline YNB13.4 gram, 10 milliliters of glycerine, 0.1 mole of potassium phosphate buffer, 0.4 milligram of vitamin H.

4. seed liquor culture condition: get an amount of thalline with the aseptic inoculation ring from above-mentioned culture dish, be inoculated in the sterilized above-mentioned seed culture medium, 20 ℃-50 ℃, the 220rpm shaking table was cultivated 12-24 hour.

5. the composition of Medium of shaking flask fermentation and every liter of content thereof are: peptone 20 grams, yeast powder 10 grams, yeast choline YNB13.4 gram, 10 milliliters of methyl alcohol, 0.1 mole of potassium phosphate buffer, 0.4 milligram of vitamin H.

6. shake flask fermentation culture condition: after the seed liquor of results Yeast engineering bacteria, centrifugal collection thalline, through the fermention medium lotion, according to volume ratio 1%-30% inoculum size Yeast engineering bacteria is inoculated in the above-mentioned Medium of shaking flask fermentation, 20 ℃-50 ℃, 100rpm-300rpm, during need the regular replenishment methanol induction keep the concentration that volume ratio is 0.5%-5%, shaking table was cultivated 1-10 days.

(3) described Human-like Collagen slightly mention checking:

The Yeast engineering bacteria fermentation ends, then centrifugal collection fermented supernatant fluid adds ammonium sulfate that mass percent is 60%-100% or sodium sulfate or sodium-chlor and precipitates concentratedly, and last centrifugal collecting precipitation obtains the Human-like Collagen crude product; Be the SDS-PAGE gel detection of 5% concentrated glue and 10%-20% separation gel again through mass percent.

Innovative point of the present invention has been to obtain a synthetic human-like collagen gene that is applicable to yeast expression system, one strain can be expressed the Yeast engineering bacteria of small molecular weight Human-like Collagen, and the alternative gelatin of the Human-like Collagen of the small molecular weight of expression is used for vaccine stabilizing agent.

The invention has the advantages that, utilize the Human-like Collagen of yeast gene engineering bacteria production not only to have good biological characteristics, and compare with the product of animal-origin have virus-free hidden danger, the good characteristic such as low rejection, so the collagen protein of purified rear alternative animal-origin is applied to vaccine stabilizing agent.

Culture presevation information:

Title: pichia spp (Pichia sP.), numbering CGMCC No.4187;

Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center;

Deposit number: CGMCC No.4187;

The preservation time: on September 20th, 2010.

The structure of embodiment 1 expression vector

Synthetic external source fragment obtains with the form that is connected on the plasmid pUC57, this plasmid and carrier for expression of eukaryon pPICZ α are carried out double digestion with EcoRI and two kinds of enzymes of XbaI simultaneously, then reclaim synthetic human-like collagen gene (referring to Fig. 1) and the linearizing carrier pPICZ α of 300bp size.

Utilize the T4 dna ligase that above-mentioned two fragments are connected, namely be configured to expression vector pPICZ α/col.Then linked system is converted into competent escherichia coli cell, utilize Sac I single endonuclease digestion method to verify 14 clone institute upgrading grains, because only containing 1 Sac I restriction enzyme site on the correct plasmid, so cutting, enzyme finishes the positive result who plasmid linearization occurs, referring to Fig. 2.And will screen 11 of acquisition ^#, 12 ^#, 14 ^#Three plasmids are sent to further sequence verification, and final comparison result shows 12 ^#With 14 ^#Plasmid contains correct, complete synthetic human-like collagen gene.

The structure of embodiment 2 Yeast engineering bacterias

Embodiment

1. the linearizing of plasmid

Expression vector pPICZ α/col needs to transform the pichia pastoris X-33 competence after the linearizing, and this purpose is in order to obtain higher transformation efficiency.In this experiment, the expression vector after the linearizing is by the rear gained that reclaims of Sac I digestion.

2. the yeast cell electricity turns competent preparation

(1) in containing the 50ml centrifuge tube of 5mlYPD, cultivate pichia spp, 30 ℃ are spent the night.

(2) get the 0.5ml overnight culture, inoculation contains the 2L shaking flask of 500ml fresh culture, and overnight growth is to OD6001.5.

(3) at 4 ℃, the centrifugal 5min collecting cell of 1500g is with the aqua sterilisa suspension cell of 500ml precooling.

(4) as above centrifugal, with the aqua sterilisa suspension cell of 250ml precooling.

(5) as above centrifugal, with the 1M sorbyl alcohol suspension cell of 20ml precooling.

(6) as above centrifugal, with the 1M sorbyl alcohol suspension cell of 1ml precooling, to the about 1.5ml of final volume.

3. transform:

(1) gets the above-mentioned cell of 80ul and mix with the linearizing expression vector pPICZ of 15ug α/col (being dissolved in 5ulTE), change in the 0.2cm electricity revolving cup of precooling.

(2) place 10min on ice.

(3) adjust the electroporation apparatus parameter: voltage 1.5KV; Electric capacity 25 μ F; Resistance 200 Ω.The electric shock sample, the electric shock time is 4.6msec.

(4) the 1M sorbyl alcohol that adds immediately the 1ml precooling is transferred to content in the centrifuge tube of 1.5ml to cup.30 ℃ of incubators were placed 2 hours.

(5) be divided into the 200ul equal portions, be applied on the YEPD solid medium flat board that contains bleomycin.

(6) hatching dull and stereotyped extremely clone at 30 ℃ produces.

Embodiment 3 recombination yeast engineering screenings:

1. bacterium colony PCR checking:

Grow mono-clonal on the YEPD flat board to be transformed, through colony polymerase chain reaction (PCR) method primary dcreening operation positive colony, again take Yeast genome as template amplification purpose fragment and send to order-checking.The final positive colony that has finally obtained 9 Yeast engineering bacterias from more than 200 transformants is referring to Fig. 3.

2. shake flask fermentation checking:

The positive colony of above-mentioned Yeast engineering bacteria also needs further to verify by the shake flask fermentation experiment expression of Human-like Collagen.Pichia pastoris X-33 is to increase the volume fermentation, utilize first seed culture medium to cultivate seed, then centrifugal collection thalline is forwarded to and continues in the Medium of shaking flask fermentation take methyl alcohol as carbon source to cultivate, adding volume ratio every 12 hours and be 0.5% methyl alcohol induces, fermentation to 96 hour end, collecting supernatant liquor, is 60% ammonium sulfate precipitation precipitation acquisition final sample through mass percent.Be the SDS-PAGE detected through gel electrophoresis Human-like Collagen of 5% concentrated glue and 15% separation gel with mass percent at last, referring to Fig. 4.Compare 3 with contrast (the yeast X-33 bacterial strain that contains blank expression vector pPICZ α) ^#Bacterial strain has band of expression at expection albumen size place (about 12.8KD).Mass percent is that the prescription of 5% concentrated glue and 15% separation gel is as shown in table 1:

Table 1:SDS-PAGE gel formula

Annotate: percentage sign is mass percent in the table.

Claims (2)

1. a Human-like Collagen that can be used for vaccine stabilizing agent is characterized in that, obtains this Yeast engineering bacteria called after pichia spp (Pichia sp.) X-33/col CGMCC No.4187 by the Yeast engineering bacteria fermentation;

Integrate a kind of human-like collagen gene col of synthetic on the described Yeast engineering bacteria karyomit(e), its base sequence is as follows:

GAAGCTGGTTTGCCTGGTGCTAAGGGTCTGACTGGTTCTCCTGGTTCTCCCGGACCCGATGGTAAGACTGGACCCCCAGGACCAGCTGGTCAAGATGGACGTCCTGGACCTCCAGGCCCTCCAGGGGCCCGAGGCCAAGCAGGTGTTATGGGGTTTCCTGGACCAAAGGGAGCAGCCGGCGAGCCAGGTAAAGCCGGCGAAAGGGGTGTCCCAGGACCACCAGGTGCTGTGGGACCAGCCGGCAAAGACGGTGAGGCAGGTGCTCAGGGTCCACCAGGACCTGCTGGTCCTGCTGGAGAAAGATAA

Its length nucleic acid is 306bp.

2. the production method of the described Human-like Collagen of claim 1 is characterized in that,

(1) structure of Yeast engineering bacteria:

According to known collagen, amino acid sequence, through the modification of yeast codon preference, finally obtain synthetic human-like collagen gene col;

Again synthetic human-like collagen gene col is cloned into Yeast expression carrier pPICZ α, called after pPICZ α/col;

Utilize electric shock transformation method that recombinant plasmid pPICZ α/col is converted into yeast cell X-33;

Utilize at last the correct Yeast engineering bacteria of method screening of PCR and order-checking;

(2) the shaking flask inducing culture of Yeast engineering bacteria:

The composition of solid medium YEPD and every liter of content thereof are: peptone 20 grams, yeast powder 10 grams, glucose 20 grams, agar powder 15 grams;

Solid culture condition: Yeast engineering bacteria is inoculated in the culture dish of above-mentioned solid medium, cultivated 3-5 days in 20 ℃-30 ℃;

The composition of seed culture medium and every liter of content thereof are: peptone 20 grams, and yeast powder 10 grams are without amino yeast nitrogen YNB13.4 gram, 10 milliliters of glycerine, 0.1 mole of potassium phosphate buffer, 0.4 milligram of vitamin H;

The seed liquor culture condition: get an amount of thalline with the aseptic inoculation ring from above-mentioned culture dish, be inoculated in the sterilized above-mentioned seed culture medium, 20 ℃, the 220rpm shaking table was cultivated 12-24 hour;

The composition of Medium of shaking flask fermentation and every liter of content thereof are: peptone 20 grams, and yeast powder 10 grams are without amino yeast nitrogen YNB13.4 gram, 10 milliliters of methyl alcohol, 0.1 mole of potassium phosphate buffer, 0.4 milligram of vitamin H;

Shake flask fermentation culture condition: after the seed liquor of results Yeast engineering bacteria, centrifugal collection thalline, resuspended through fermention medium, the 1%-30% inoculum size of the resuspended volume of Yeast engineering bacteria is inoculated in the above-mentioned Medium of shaking flask fermentation, 20 ℃, 100rpm-300rpm, during need regular replenishment methyl alcohol to induce, and keep the 0.5%-5% that methanol concentration is volume ratio, shaking table was cultivated 1-10 days;

(3) Human-like Collagen slightly mention checking:

The Yeast engineering bacteria fermentation ends, then centrifugal collection fermented supernatant fluid adds ammonium sulfate that mass percent is 60%-100% or sodium sulfate or sodium-chlor and precipitates concentratedly, and last centrifugal collecting precipitation obtains the Human-like Collagen crude product; Be the SDS-PAGE gel detection of 5% concentrated glue and 10%-20% separation gel again through mass percent.

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