CN103173474B - Gene sequence for expressing soluble recombinant human hyaluronidase PH20 in CHO (Chinese hamster ovary) cell - Google Patents

Abstract

The invention discloses a gene sequence for expressing soluble recombinant human hyaluronidase PH20 by a CHO (Chinese hamster ovary) cell. The gene sequence has a nucleotide sequence shown in SEQ ID NO.2; after a kozak consensus sequence is added to 5' terminal of the optimized gene sequence, the optimized gene sequence is connected to pOptiVEC-TOPO carrier, and stably transferred to the Chinese hamster ovary cell improved by genetic engineering, so as to achieve high expression of the soluble recombinant human hyaluronidase PH20 in a mammalian cell.

Description

A kind of gene order for expressing cho cell soluble recombined human Unidasa PH20

Technical field

The invention belongs to biological technical field, be specifically related to a kind of for the gene order of Chinese hamster ovary cell (Chinese hamster ovary celI) recombinant expression of soluble Unidasa and the engineering cell strain that contains this gene.

Background technology

Unidasa is the hyaluronic enzyme family of degraded, and hyaluronic acid is the essential component of extracellular matrix and the main component of interstitial barrier.Unidasa can make hyaluronic acid produce degraded effect, thereby reduces hyaluronic activity and viscosity, improves liquid infiltration ability in tissue.

Hyaluronic acid enzyme family comprises bacterium Unidasa, from the Unidasa of parasite and crustacean with from mammiferous Unidasa.And in people's genome, there are 6 hyaluronic acid-like enzyme genes: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20/SPAM1.Wherein, HYAL3, HYAL4 present not to be had or little degraded hyaluronic acid activity, and HYAL1, HYAL2 are acid activity Unidasas, the general catalytic activity that lacks under condition of neutral pH, PH20 is neutral organized enzyme, within the scope of the blood of human body or the pH of tissue juice, has catalytic activity.

Unidasa is mainly used to improve the infiltration of other injectable drugs and increases the tissue permeability of other drug and promote diffusion or disperse.Modal application is the rapid osmotic (as dentistry) of ophthalmologic operation and local anesthetic, cannot find blood vessel time, uses in child vein drop.Also can be used for treating tumour, coordinate subcutaneous injection to replace intravenous drip with expensive antibody, keep the purposes such as unobstructed of Regular Insulin and other pump administration.Owing to deriving from, the Unidasa immunogenicity of extracting in animal tissues is stronger, is unsuitable for applying to human clinical, and utilizing genetically engineered recombinant technology to express, produce people's Unidasa has become inevitable trend.Because the C end of people's total length PH20 aminoacid sequence contains glycosyl-phosphatidyl inositol (GPI) decorating site, affect its solubility secretion outside host cell, hold corresponding encoding sequence therefore will remove PH20 precursor protein (precursor) aminoacid sequence C while building the strain of solubility PH20 secreting, expressing, and leave encoding sequence corresponding to 1-482 amino acids in PH20 precursor protein.The albumen of expressing is called soluble recombined human PH20 albumen (soluble rHuPH20).As the Hylenex Unidasa of U.S. FDA approval listing, be soluble rHuPH20.

Chinese hamster ovary cell (Chinese hamster ovary cell, CHO) is a kind of fibroblast that derives from Chinese hamster ovary, is the host cell that mammalian expression system is conventional.It has lot of advantages: post transcriptional modificaiton function accurately, the glycosylation pharmaceutical protein of expressing is approaching native protein molecule most aspect molecular structure, physicochemical property and biological function, expression product cell exocrine, be convenient to separation and purification, and because it belongs to inoblast, seldom secretion intrinsic protein, utilizes the separation and purification of foreign protein.

We find under study for action, use the level of the engineering cell strain expression PH20 building containing the genetically engineered Chinese hamster ovary celI strain (being specially DG44 cell strain) of carrier transfection of solubility rHuPH20 natural gene sequence lower, cause production cost high.We can improve the expression level of target protein in Chinese hamster ovary celI strain greatly at the gene order after to native sequences optimization.

Summary of the invention

The object of the invention is to: for the defect of the low expression of natural gene of coding solubility rHuPH20, by optimized gene sequence, realize the high expression level of solubility rHuPH20 in CHO engineering cell strain.

The technical solution used in the present invention is:

The gene order of the people's Unidasa PH20 optimizing, it has the nucleotide sequence shown in SEQ ID NO.2.

Add kozak consensus sequence at above-mentioned people's Unidasa PH20 optimized gene sequence 5 ' end, obtain its derivative DNA sequence dna

A kind of cloning vector, contains above-mentioned people's Unidasa PH20 optimized gene sequence or by its derived dna sequence.

A kind of expression vector, contains above-mentioned people's Unidasa PH20 optimized gene sequence or by its derived dna sequence.

The initial vector of described expression vector is mammalian cell expression vector.

A kind of Chinese hamster ovary cell expression strain, contains above-mentioned expression vector.

Described Chinese hamster ovary cell expression strain is DHFR gene defection type.

Above-mentioned Chinese hamster ovary cell expression strain is in the application of producing on soluble recombined human Unidasa PH20.

The invention has the beneficial effects as follows: the gene order of the PH20 of the present invention after optimizing, be connected to pOptiVEC-TOPO carrier and complete plasmid construction, be stably transfected into after genetically engineered Chinese hamster ovary cell, significantly improve soluble recombined human Unidasa PH20(soluble rHuPH20 with former sequence phase specific energy) expression level.

Brief description of the drawings

Fig. 1 is the solubility rHuPH20 gene order optimized of the present invention and the aminoacid sequence comparative analysis of natural gene sequence encoding;

Fig. 2 is pOptiVEC-TOPO carrier structure schematic diagram.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.

Due in operation below by relate to the detection of hyaluronidase activity and PH20 gene copy number quantitatively, so first introduce the operation of these two kinds of methods here.

One, the activity test method of Unidasa (nephelometry)

1, nephelometry principle: hyaluronic acid (HA) solution of acidifying can form stable colloidal solution with serum, after hyaluronic acid decomposes, will become clarification with the mixed solution of serum.Because Unidasa has the effect that catalysis hyaluronic acid decomposes, therefore within the specific limits, hyaluronidase activity and reacted turbidity are inversely proportional to.

2, material: horse serum and HA reference substance are all from SIGMA company.HA is configured to 0.5U/mL, 1U/mL, 2U/mL, 4U/mL, 6U/mL, 8U/mL, 10U/mL totally 7 activity unit's reference substances;

3, concrete testing process: HA solution adds in 96 orifice plates, and every hole 20 μ L, then add 20 μ L reference substance or sample liquid, fully mixes, 37 DEG C of water-bath 30 min, and after taking out, every hole adds 160 μ L horse serums, mixes, and room temperature is placed 30 min, surveys a ₆₄₀.With absorbancy afor ordinate zou, tire (U) of reference substance solution is X-coordinate, drawing standard curve.The sample absorbancy recording calculates activity according to typical curve.

Two, express the quantitative PCR detecting method of solubility rHuPH20 gene copy number in strain

1, design of primers and synthetic

Design respectively primers F 1/R1, F2/R2 according to people's Unidasa PH20 gene order natural and that optimize, according to mouse house-keeping gene β-actin gene order design primer β-actin F/R, in table 1.

2, extract CHO DG44 genomic dna and recombinant plasmid

Extract each recombinant cell strain genome, CHO DG44 genomic dna and recombinant plasmid, by DNA quality and the concentration of the Detection and Extraction of nucleic acid-protein analyser.

3, standard substance build

Calculating contains 1,5,25,125,625 required expression vector quality of copy number of foreign gene, and CHO DG44 genome quality is got 10ng, and formula is as follows:

[copy number × containing the expression vector size (N bp) of PH2O gene]/[CHO DG44 group DNA single times body size (3 × 10 ⁹bp)]=containing transgene expression vector quality/CHODG44 genome quality

The expression vector of different mass (representing different copy numbers) is mixed with CHO DG44 genomic dna (10 ng), build standard substance.

4, determining of typical curve

Turn PH20 gene fragment with PH2O primers F/R amplification, the amount using β-actin F/R amplification house-keeping gene β-actin as internal reference mark genomic dna.The amplified production of PH20 F1/R1 primer is 445 bp; The amplified production of PH20 F2/R2 primer is 445 bp; The amplified production 200bp of β-actin F/ R primer.PH20 gene fragment detection primer amplification Ct (PH20) is deducted to the amplification Ct (β-actin) of corresponding β-actin gene, the △ Ct obtaining, then the mapping of the logarithmic value of copy number to sample obtains the typical curve of absolute quantitation.

5, real-time quantitative PCR reaction system and condition

Reaction system is: template 2 μ L, upstream primer (10 pmolL ^-1) 0.8 μ L, downstream primer (10 pmolL ^-1) 0.8 μ L, SYBR Green Real-Time PCR Mix 10 μ L, distilled water 6.4 μ L.

Reaction conditions is: 95 DEG C of 10 min; 95 DEG C of 10 s, 60 DEG C of 20 s, 72 DEG C of 30 s, 32 circulations.Each sample arranges 3 Duplicate Samples, gets average.

6, recombinant cell strain solubility rHuPH20 gene copy number qualification

Extracting each recombinant cell strain genome, is 0.8% agarose gel electrophoresis and A260/A280 ultraviolet detection through volume fraction, shows that genome pollutes without protein and RNA.Get respectively 10 ng and carry out real-time quantitative PCR, amplification PH20 gene and β-actin gene, and independently repeat experiment for 3 times, draw the △ Ct value of each recombinant cell strain about PH20 gene.Copy number calculates according to formula corresponding to typical curve.

Embodiment

One, the optimization of the gene order of soluble human hyaluronidase enzyme PH20

On the basis of analyst's Unidasa PH20 natural gene sequence (nucleotide sequence is as shown in SEQ ID NO.1), eliminate rare codon, remove unstable motif, removal RNA loop-stem structure and utilize the best codon of Chinese hamster ovary cell to redesign.Gene order after optimization is as shown in SEQ ID NO.2.Gene order and native sequences after optimizing are carried out to nblast homology and compared, do not show that both have the result of homology; And carry out homology comparison with tblastx, show the aminoacid sequence 100% homology (see figure 1) that both encode.

For improving the expression efficiency of this gene order in eucaryon host, at gene order coded amino acid position 1(methionine(Met)) add Kozak consensus sequence GCCACC before corresponding codon.Gene order after optimization is synthetic by English Weihe River company of prompt base (Shanghai) trade Co., Ltd.

The screening of the height copy/overexpression cell line that two, comprises optimized gene

1, the structure of carrier for expression of eukaryon (PH20-pOptiVEC – TOPO)

With PCR primers F 2 and R2(in table 1) increase PH20 optimized gene synthetic (before coding region containing kozak sequence), the gene fragment of amplification is connected to carrier for expression of eukaryon pOptiVEC-TOPO(by T-A cloning and sees Fig. 2) in, build and obtain recombinant expression vector PH20-pOptiVEC – TOPO.

2, recombinant expression vector transfection CHO-DG44 cell

Electroporation transfection CHO-DG44 cell for PH20-pOptiVEC – TOPO recombinant vectors.Wherein, CHO-DG44 cell used is DHFR gene defection type, does not contain dihydrofolate reductase gene, can not nucleic acid, must in the substratum that contains xanthoglobulin (H) and thymidine (T), grow.

Screening positive cell principle is: in the time that the goal gene of transfection is connected with dhfr gene, positive cell has also just obtained dhfr gene.Can in the substratum that lacks HT, grow.

Transfection process is as follows:

With CD OptiCHO culture medium culturing amplification CHO DG44 cell, before transfection, carry out cell counting, guarantee that 95% for viable cell.

Centrifugal collection 6 × 10 ⁷cell precipitation, and at 2 × transfection damping fluid (40mM HEPES, pH7.0,274mM NaCl, 10Mm KCl, the 1.4mM Na of 0.7mL ₂hPO ₄, 12mM dextran) in resuspended one-tenth 2 × 10 ⁷the density of cell.Add 90 μ L(250 ug thereafter) use claIthe recombinant plasmid of restriction endonuclease linear process, and cell/plasmid solution is transferred in electroporation cuvette in room temperature.The mixture of cell/plasmid carries out electroporation processing under the condition of the electrical condenser of 875V/cm and 960uF energising.

3, the cell of screening stable transfection

In order to obtain the cell strain of high expression level Unidasa PH20,, by the successful cell of a collection of stable transfection of screening, successfully insert cell chromosome with the pOptiVEC – TOPO Vector of goal gene.Process is as follows:

After electroporation, cell is shifted out from cuvette and transfer in the CD OptiCHO substratum of 5mL, and make them in 6 porocyte culture plates, in 5% CO ₂in 37 DEG C in the incubator of humidification without the growth 2 days of selecting.After 2 days, get supernatant and detect Unidasa content with nephelometry.Collecting cell, counts and uses the CD OptiCHO substratum of xanthoglobulin (H), thymidine defect (T) to be diluted to 2 × 10 ⁴individual viable cell/mL.Cell suspension is transferred to 96 porocyte culture plates by 0.1mL/ hole, adds H, T defect substratum to 0.2mL.Then at 5%CO ₂, cultivate 3-4 days in the incubator of 37 DEG C.Collect every hole supernatant nephelometry and detect Unidasa content.Select 5 clones that expression amount is the highest, increase with the substratum of H, T defect, after collecting cell, be settled to 5 × 10 with CD OptiCHO substratum ⁵individual viable cell/mL, and frozen processing.

4, with Rheumatrex (MTX) amplification, the high copy/high expression level monoclonal cell of screening

(1) MTX carries out gene amplification

The operation of carrying out gene amplification with MTX is as follows:

After freeze-stored cell recovery, carry out amplification cultivation with CD OptiCHO substratum, centrifugally remove old substratum, with 3 × 10 ⁵/ mL cell density is inoculated in the CD OptiCHO substratum that 100-300mL contains 50nM MTX.Enchylema is transferred in the culturing bottle of 0.5-1.0 L, in 5%CO ₂, culturing cell in the incubator environment of 37 DEG C.Every 3-4 days passage cell is in fresh substratum, and inoculum density is 2 × 10 ⁵-3 × 10 ⁵cell/mL.In the time that active cells number starts to increase, start passage cell, inoculum density is 2 × 10 ⁵-3 × 10 ⁵cell/mL.In the time that viable count is greater than 90%, freeze-stored cell.

(2) limiting dilution assay screening cell clone

In limiting dilution assay, cell substratum used is for cloning substratum completely, from Invitrogen company.100mL clones composition in substratum completely: 86mL CD FortiCHO; The freshly prepared 200mM L-glutamine of 3mL; 10mL conditioned medium; 1mL 100 × HT mother liquor.

Limiting dilution assay operation steps is as follows:

1. prepare the centrifuge tube of several 50mL, accurate calculation cell density, the cell of getting 5-10mL stable transfection is added to centrifuge tube 1, then doubling dilution to 1000 cell/mL.

2. substratum is cloned in preheating completely, draws 200 μ L cell suspensions (1000 cells/mL) to cloning completely in substratum from dilution tube.Reverse 5-6 time back and forth, transfer to sterile chamber.The cell that is divided into 200 μ L dilutions is in culture plate in 96 orifice plates.Under 37 DEG C of conditions, cultivate 96 orifice plate 10 days.After 10 days, clone with microscope observing cell.Nephelometry detects cell expressing situation, and the expression amount detecting and copy number results, in table 2, filter out 5 high-expression clone cell strains.As known from Table 2, under the condition of similar copy number, the expression amount that the PH20 gene pairs of optimizing in the present invention is answered will be significantly higher than the expression amount that natural PH20 gene pairs is answered.

Note: ¹for compared with natural PH20 group, expression amount difference is (p<0.01) very significantly

Above embodiment is only for introducing preferred case of the present invention, and to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention, all should be regarded as a part of the present invention.

<110> Guangzhou Baiyunshan Baidi Biotechnology Co., Ltd.

<120> gene order for expressing cho cell soluble recombined human Unidasa PH20

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<160> 8

<170> PatentIn version 3.5

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cctcctgtta ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180

cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga 240

ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg ctactatcct 300

tacatagatt caatcacagg agtaactgtg aatggaggaa tcccccagaa gatttcctta 360

caagaccatc tggacaaagc taagaaagac attacatttt atatgccagt agacaatttg 420

ggaatggctg ttattgactg ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480

aaagatgttt acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540

ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg 600

gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg ttattatctt 660

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cgagttcggg aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900

tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa 960

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agagtgcgcg aggccatcag agtgtccaag atccccgacg ccaagtcccc cctgcctgtg 900

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ctggtgtata ccttcggcga gacagtggcc ctgggcgcct ctggaatcgt gatctggggc 1020

accctgtcca tcatgcggtc catgaagtcc tgcctgctgc tggacaacta catggaaaca 1080

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Claims (8)

1. the gene of the people's Unidasa PH20 optimizing, its nucleotide sequence is as shown in SEQ ID NO.2.

2. by the derivative DNA of the optimized gene of claim 1, it is characterized in that, add kozak consensus sequence at optimized gene 5 ' end.

3. a cloning vector, it contains gene claimed in claim 1 or DNA claimed in claim 2.

4. an expression vector, it contains gene claimed in claim 1 or DNA claimed in claim 2.

5. expression vector according to claim 4, is characterized in that, initial vector is mammalian cell expression vector.

6. a Chinese hamster ovary cell expression strain, it contains the expression vector described in claim 4 or 5.

7. Chinese hamster ovary cell expression strain claimed in claim 6, it is DHFR gene defection type.

8. Chinese hamster ovary cell expression strain claimed in claim 6 is in the application of producing on soluble recombined human Unidasa PH20.