CN105713882A - Purification method of recombinant sendai viruses - Google Patents

Abstract

The invention mainly relates to a purification method of recombinant sendai viruses, which is suitable for the large-scale preparation of recombinant sendai virus-vectored vaccines. The purification method is characterized by comprising the following steps: vacuum filtration, affinity chromatography, ultrafiltration, nuclease treatment and gel filtration chromatography. The purification method has the advantages that the operation is simple, the quality is stable, the yield is high, etc.

Description

A kind of purification process of recombinant sendai virus

Technical field

The invention belongs to biomedicine technical field, relate to a kind of Sendai virus purification process meeting recombinant sendai virus vector vaccine large-scale production.

Background technology

Sendai virus gene group is a strand RNA, and length is 15384 nucleotide, includes 6 genes altogether.The big subunit L (largeprotein) of NP (nucleoprotein), phosphorylated protein P (phosphoprotein), polymerase and RNA collectively form nucleoprotein complex RNP.Matrix protein (matrixprotein) is for the assembling of virion.Hemagglutinin neuraminidase HN (hemagglutinin-neuraminidase) and fusion protein F (fusionprotein) are in the glycoprotein on virus after birth, are primarily involved in the process such as absorption and intrusion of virion and host cell.

Leader (ld) and trailer (tr) is distinctive targeting sequencing and end sequence in Sendai virus gene group respectively.Six genes are from beginning to end to be arranged in genome, each gene expresses unit (open reading frame) as one, wherein can be divided into again several independent region, being gene initiation region GS (GeneStart), not transcript regions UTR (Untranslateregion) and gene end region GE (GeneEnd) respectively, I (intergenicmotif) is the junction that the 3bp fragment repeated is arranged in gene and gene.

The packaging process of Sendai virus: after pSeV plasmid enters cell, at helper virus vTF7-3 (Fuerst, T.R.etal, Proc.Natl.Acad.Sci.USA83:8122-8126,1986) start to replicate positive chain RNA under the effect of T7 polymerase, it is copied into strand RNA under the effect of N, P and the L albumen of helper plasmid pTM-N, pTM-P and pTM-L synthesis, namely Sendai virus gene group, then start to express M albumen, HN albumen, F protein, and it is finally packaged as complete virion, to sprout, form forms the Sendai virus with infection ability.

Development along with reverse Genetics Technique, Sendai virus is developed to kytoplasm and expresses the viral vector propagating defective, preclinical study has proven to its foreign gene-carrying to some disease, has good therapeutical effect such as respiratory tract disease, ischemia injury and tumor etc..

Owing to Sendai virus belongs to negative single strand RNA virus, all biocycle such as the duplication of virus and assembling all completes in Cytoplasm, also without DNA building-up process during duplication, and synthesis and the transcription and translation of positive and negative chain RNA is only carried out with itself for template, still do not find that it has the mechanism and sign being integrated in nucleus DNA, therefore use bigger safety guarantee, solve the safety issue produced in gene therapy for a long time；Secondly as this outer virionic membrane contains F protein specific to Sendai virus, therefore efficiently its RNP can be proceeded to cell, replicate and produce enough destination protein products after transcribing, reach the purpose for the treatment of；Further, since its efficiency of infection is high, using dosage is little, and the side effect therefore itself caused by virus in therapeutic process is more much smaller than other carrier system, and safety is higher；It has now been discovered that Sendai virus can infect the almost all kinds of cell including myocyte, neurocyte and epithelial cell, this feature makes its application surface widen further, can be widely used in the every field such as gene therapy and genetic immunization.

In preclinical study, recombinant sendai virus vector all shows fabulous therapeutic effect in severe lower limb ischemia gene therapy formulation, Alzheimer (degenerative brain disorder) vaccine and AIDS vaccine.

Recombinant virus before application, after will breeding, cracks from host cell and is purified in host cell.Select a kind of suitable purification process most important to obtaining highly purified recombinant virus.

The present invention provides a kind of recombinant sendai virus purification process, and the method can meet large-scale production recombinant sendai virus vector vaccine, has established theoretical basis for the safe and effective recombinant sendai virus vector vaccine of large-scale production.

Summary of the invention

The purification process of a kind of recombinant sendai virus, it is characterised in that comprise the following steps:

(1) the initial liquid of vacuum filter virus

(2) affinity chromatograph

(3) ultrafiltration

(4) nuclease processes

(5) gel permeation chromatography；

(6) measure virus titer and carry out SDS-PAGE electrophoresis.

Wherein, the initial liquid of vacuum filter virus selects 0.45 μm of membrane filtration.

After membrane filtration, the filtrate comprising virus is carried out affinity chromatograph.Affinity chromatograph is preferably used sulfonated cellulose to carry out.This chromatography media aglucon is sulfonic group polysaccharide, it is possible to combine with Sendai virus surface HN protein-specific, under low ionic strength can adsorb major part Sendai virus, and can be eluted under high ionic strength, simultaneously that virus activity impact is little.The virus-culturing fluid collected directly goes up affinity column after diluting with the phosphate buffer of low ionic strength, and the phosphate buffer of low ionic strength is preferably containing 75mMNaCl solution.After rinsing pillar with the phosphate buffer of low ionic strength, then the phosphate buffer eluting with high ionic strength, it is preferred to the phosphate buffer containing 1MNaCl, collect eluent.

Then being concentrated by ultrafiltration by elution of virus liquid, the preferred molecular cut off of ultrafilter membrane bag is the ultrafilter membrane bag of 500K.

Concentrated solution is processed with nuclease, to remove the host DNA impurity in concentrated solution after ultrafiltration concentration.

Nuclease of the present invention is Benzonase.

Nuclease proceeds gel filtration after processing virus liquid, and solvent resistant column is preferably Sepharose4FF chromatographic column, collects virus peak and is the whole liquid of virus.

The whole liquid titer determination of virus adopts hemagglutination test (HA) method and cell infection unit (cellinfectiousunit, CIU) method.

HA algoscopy: Sendai virus outer membrane face exists the HN albumen can being combined with erythrocyte surface sialic acid residues, this albumen has sialidase activity concurrently simultaneously, sialic acid residues on cleavable cell surface glycoprotein, but what HN main manifestations went out at low temperatures is sialic combination activity, therefore can be used to measure the Hemagglutination titer of virus.Within the specific limits, may occur in which complete blood clotting when measuring in hole cell density less than or equal to virus concentration, once virus numbers is lower than cell number, then incomplete blood clotting occurs.Therefore carrying out dripping the hole that can will appear from complete blood clotting when poison measures and be decided to be blood clotting terminal, the virion subnumber in this hole is equal with cell number, thus can calculate the density of virion in sample.

As shown in Figure 1, the complete coagulation person of blood cell is positive (all black circle), and the virion density of the whole liquid of virus of the purification process purification of the present invention is: 8 × 10⁷× 2048=1.64x10¹¹VP/ml

CIU assay method: the virus liquid of purification is non-propagating type defective virus, it is possible to host cells infected LLC-MK2, replicates wherein and produces the progeny viral particles without infection ability.Metainfective cell determines activated virion number in test sample by Fluorescent microscope counting after dyeing with indirect immunization.Employing CIU measures the virus just liquid that just cracking has obtained from cell and the titre of the viral whole liquid obtained through purification process purification of the present invention respectively, the titre of virus just liquid is 2.31E+11, the titre of the whole liquid of virus is 1.45E+11, reaches 62.45% with the purification efficiency of the purification process of the present invention.

Meanwhile, the whole liquid of the virus of purification is also carried out SDS-PAGE electrophoresis by the present invention, and result can be observed typical 7 band of Sendai virus.

Accompanying drawing explanation

Fig. 1 is shown that the recombinant sendai virus SDS-PAGE result that purification of the present invention obtains, and swimming lane 1 represents molecular weight standard (being purchased from Promega), and swimming lane 2 represents the recombinant sendai virus of purification.

Fig. 2 is shown that HAU method and surveys virus titer result figure, and completely black circle represents the complete coagulation of blood cell, the incomplete coagulation of representative a little or not coagulation in circle.

Detailed description of the invention

Embodiment 1

The initial liquid of vacuum filter virus

1. being filtered under vacuum by the Sendai virus supernatant 3000ml of collection, filter sizes is 0.45 μm；

2. collect filtrate, by the phosphate solution of filtrate 10mMpH7.2 dilution 2 times to 6000ml.

Embodiment 2

Affinity chromatograph

1. washing affinity column with 400ml phosphate buffer (comprising 1.5MNacl solution), maintenance flow velocity is 37ml/min；

2. with 400ml distilled water wash affinity column at 4 DEG C；

3., under 4 DEG C of conditions, clean chromatographic column with 400ml0.1MNaOH solution；

4. with 400ml distilled water wash affinity column at 4 DEG C；

5. with the circulating water chromatographic column in column jecket, then with ice-cold wash solution (phosphate buffer containing 75mMNaCl) washing；

6. the virus liquid loading being filtrated to get；

7. with wash solution washing affinity column ice-cold for 650ml；

8. with eluent (phosphate buffer containing the 1MNacl) eluting that 360ml is ice-cold；

9. collecting the eluent at wavelength 280nm peak value place, volume is about 150-200ml.

Embodiment 3

Ultrafiltration

1. clean ultrafiltration system with the NaOH of 500ml0.1M, then with the distilled water of same volume and phosphate buffer DPBS (without Ca²⁺/Mg²⁺) washing；

2. the pressure regulating compression pump is 20psi, and opens compression pump；

3. collect the virus liquid after ultrafiltration；

4. closing presure pump, adds 180ml5mMMgCl in the vial₂/ DPBS solution；

5. repeat step 2-3, collect virus liquid.

Embodiment 4

Nuclease processes

Being added by nuclease in the virus liquid after ultrafiltration, the concentration making nuclease is 100U/ml, 37 DEG C of digestion 30min

Embodiment 5

Gel filtration

1., with 4 DEG C of circulating chilled water cooling gel Filter columns, and with 500ml4 DEG C of distilled water wash pillar, maintenance flow velocity is 5ml/min；

2. cleaning chromatographic column with 500ml0.1MNaOH solution, 4 DEG C overnight；

3. balance chromatographic column with DPBS ice-cold for 1000ml；

4. loading；

5. with the DPBS eluant solution virus liquid that 1000ml is ice-cold；

6. collect first eluting peak on ice, with the filtering with microporous membrane elution of virus liquid of 0.22 μm；

7. virus liquid is packed as 10ml/ bottle, lowers the temperature rapidly with dry ice/ethanol, and-80 DEG C of preservations.

Embodiment 6

Virus titer measures

6.1HA algoscopy

1. measuring samples is taken out from-80 DEG C of refrigerators, be immediately placed in 37 DEG C of water-baths, treat that ice melts front taking-up completely, be put in 4 DEG C of refrigerators stand-by.

2. take orifice plate of the U-shaped end 96 and be placed in Biohazard Safety Equipment, add PBS50ul/ hole from the first hole, totally 16 holes.

3. take measuring samples 50ul and be added in the first hole, repeatedly mix with sample injector, then take 50ul and be added in next hole, be diluted to the 15th hole so successively, the 15th hole is taken out 50ul and discards；16th hole is set to negative control.

4. in each hole, add freshly prepared chicken red blood cell, 8 × 10⁷Cells/ml, 50ul/ hole.Order is for first adding negative hole (the 16th hole), then complys with rare to dense order addition chicken red blood cell, cover lid, pats plank side gently, so as to mix.

Place 1 hour for 5.4 DEG C, observed result, photograph, record.

6. result judges: the complete coagulation person of blood cell is judged to the positive, and coagulation terminal is the hole of last complete coagulation, and virus titer calculates as follows:

The viral dilution in virus titer=coagulation terminal hole

Virion density=8 × 10⁷× virus titer

6.2CIU algoscopy

1. with 2 × 10⁶/75cm²Culture bottle inoculation LLC-MK2 cell, after cultivating three days, peptic cell, in first 72 hours inoculating cells of test in six orifice plates.

2. measuring samples is taken out from-80 DEG C of refrigerators, be immediately placed in 37 DEG C of water-baths and shake, treat that ice is about to melt front taking-up completely, be put in 4 DEG C of refrigerators stand-by.

3. take aseptic EP pipe 7, respectively labelling.According to the form below adds viral dilution liquid in each pipe, and with reference in the following manner, the sample DPBS (pre-cooling) containing 1%BSA is diluted 2 × 10⁶Times.

Guan Hao	1	2	3	4	5	6	7
								Diluent volume (μ l)	270	900	900	900	900	900	500
Application of sample amount	30	100	100	100	100	100	500
								Extension rate	10	102	103	104	105	106	2×106

4. taking out six orifice plates, microscopy confirms that cell covers with, and form is good, meets requirement of experiment.

5. sucking culture fluid, every hole adds 1mlDPBS and washes one time, blots DPBS；

6. every hole adds No. 7 each 100 μ l of pipe sample, shake up, put 37 DEG C, the CO of 5%₂Incubator is cultivated 1 hour；

7. suck viral supernatants, add DPBS and wash one time, blot DPBS；

8. add the MEM of 2ml, 37 DEG C, the CO of 5%₂Incubator is cultivated 48 hours；

9. fixing: to take out 6 orifice plates, suck culture fluid, wash one time with DPBS, blot, every hole adds the methanol of 1mlDPBS (37 DEG C of temperature baths) and 1ml-20 DEG C of pre-cooling, after pre-fixing 1min under room temperature, sucks and pre-fix liquid.Add the methanol of 1ml-20 DEG C of pre-cooling again to every hole, fix 10 minutes for 4 DEG C, suck methanol, then wash one time with DPBS (1ml/ hole), the air-dry 5min of room temperature, stand-by.

10. add primary antibodie: by cell in hole, after DPBS (37 DEG C of temperature baths, 1ml/ hole) moistening, to throw away, every hole adds the anti-Sendai virus antibody of 0.5ml rabbit, cultivates 1 hour for 37 DEG C；

11. it is anti-to add two: sucks primary antibodie solution in hole, with DPBS-0.1%TritonX-100 hole flushing 3 times, 1ml/ hole, hatches 3 minutes every time.Sucking-off DPBS-0.1%TritonX-100.Every hole adds the goat anti-rabbit igg of 0.5mlFITC labelling, cultivates 1 hour, encases lucifuge with masking foil during cultivation for 37 DEG C,；

12. washing: suck two anti-solution in hole, with DPBS-0.1%TritonX-100 hole flushing 3 times, 1ml/ hole, hatch 3 minutes every time；

13. get rid of DPBS-0.1%TritonX-100 in hole, every hole adds 1mlDPBS, encases lucifuge with masking foil.

14. microscopic counting: counting the cell number having fluorescence in each hole under 5 × object lens respectively, each cell having fluorescence represents a CIU, calculates the CIU concentration of sample as follows:

Sample CIU concentration=A × 10 × 2 × 10⁶(CIU/ml)

Wherein, A is the cell average having fluorescence in 6 holes.

Embodiment 7

SDS-PAGE electrophoresis

Adopting 10%SDS-PAGE method to carry out, application of sample amount is 5.0 × 10⁸After CIU, SDS-PAGE electrophoresis, with coomassie brilliant blue staining, decolouring, it is judged that whether contain 7 characteristic bands of Sendai virus after electrophoresis.Experimental result is as it is shown in figure 1, after SDS-PAGE electrophoresis, can be observed typical 7 band of Sendai virus.

Claims (6)

1. the purification process of a recombinant sendai virus, it is characterised in that comprise the following steps:

(1) the initial liquid of vacuum filter virus；

(2) affinity chromatograph；

(3) ultrafiltration；

(4) nuclease processes；

(5) gel permeation chromatography；

(6) measure virus titer and carry out SDS-PAGE electrophoresis.

2. the purification process described in claim 1, it is characterised in that described affinity chromatograph selects sulfonated cellulose to carry out, and the phosphate buffer of low ionic strength is 75mMNaCl solution, and the phosphate eluent of high ionic strength is 1MNaCl solution.

3. the purification process described in claim 1, it is characterised in that the ultrafilter membrane bag of ultrafilter membrane bag to be molecular cut off be 500K in described ultrafiltration.

4. the purification process described in claim 1, it is characterised in that described nuclease is Benzonase.

5. the purification process described in claim 1, it is characterised in that the Filter column of described gel permeation chromatography is Sepharose4FF post.

6. the application in recombinant sendai virus vector production of vaccine of the purification process described in claim 1.