CN1686541A

CN1686541A - Preparation method of human macrocell virus pp65 protein vaccine

Info

Publication number: CN1686541A
Application number: CN 200510038861
Authority: CN
Inventors: 王明丽
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-04-11
Filing date: 2005-04-11
Publication date: 2005-10-26

Abstract

A human cytomegalovirus pp65 protein vaccine for preventing and blocking primary HCMV infection is prepared through extracting HCMV DNA, amplifying it to obtain the gene fragment pp65(1006-152/nt), cloning it to the prokaryotic expression carrier PET100-TOPO, exogenous target gene expression in E.coli BL21 cell, extracting target fragment protein pp65, and purifying.

Description

The preparation method of human macrocell virus pp 65 protein vaccine

Technical field

The present invention relates to a kind of protein biological preparation, is a kind of be used to prevent parent constitutional or infection of activity Human Cytomegloviru (HCMV), the vaccine of blocking-up HCMV vertical transmission.

Background technology

Human cytomegalic inclusion disease virus (Human Cytomeglalovirus) HCMV belongs to the distrand DNA virus of herpesvirus β subfamily, cmv infection is very extensive among the crowd, primary infection is inapparent infection usually, minority has clinical symptoms, the existing CMV antibody of 60-90% adult, though produce specific antibody but most of people infects the back, transferred to long-term band poison from the human body removing does not become latent infection to virus.The gravid woman that former or activity HCMV infect is being taken place, intrauterine infection can take place in addition cause miscarrying, sequela such as deafness that fetal anomaly and cerebral nervous system infringement are left over is blind.Congenital HCMV infects the vertical transmission that mainly comes from parent, and the HCMV primary infection that therefore must can effectively prevent parent before infringement takes place is that the blocking-up congenital HCMV infects the key that takes place.

In recent years two kinds of HCMV attenuated live vaccines (AD169 and Townel 125) of external development are tested in healthy volunteer and organ transplantation person; but this vaccine protective capability is limited; and can not get rid of the possibility that activation and canceration take place in the body because virus is hidden for a long time; having the scholar to extract HCMV subunit peptide subsequently carries out in animal experiment and the human trial as antigen; discovery by this boosting vaccine body, induce generation neutralizing antibody all very limited on amount and time; and the extraction process complexity costs an arm and a leg.

Summary of the invention

The preparation method of HCMVpp65 protein vaccine, be to utilize molecular biological technology and method, extract HCMV DNA earlier, and be pp65 (1006-1521nt) genetic fragment that template amplification goes out to have CTL and bone-marrow-derived lymphocyte antigen advantage epi-position with it, be cloned into the PET100-TOPO prokaryotic expression carrier, and in the E.coliBL21 cell, carry out the expression of external source genes of interest, identify its specificity and immunogenicity after, a large amount of extraction and purification HCMVpp65 purpose fragment protein.The HCMVpp65 protein vaccine can break parent physiologic immunity tolerance status and obtain stronger, specific humoral immunization and cellular immunization, thereby blocking-up HCMV congenital infection obtains the excellent protection effect in inducing the pregnant women organism immune response.

The preparation method of human macrocell virus pp 65 protein vaccine, it is characterized in that extracting HCMV DNA, and be pp65 (1006-1521nt) genetic fragment that template amplification goes out to have CTL and bone-marrow-derived lymphocyte antigen advantage epi-position with it, be cloned into the PET100-TOPO prokaryotic expression carrier, and in E.coli BL21 cell, carry out the expression of external source genes of interest, identify its specificity and immunogenicity, a large amount of extraction and purification pp65 purpose fragment protein.

The pp65 protein vaccine only is used in mouse model at present: what the present invention selected in the experiment is that 30 of Balb/c mices (SPF level), heavily about 18-20g are divided into five groups, six every group at random:

A group (pp65 protein vaccine immune group) every mice is subcutaneous multi-point injection 50 μ gTpp65 albumen through the back respectively for the first time, the subcutaneous multiple spot booster injection 50 μ gTpp65 albumen through the back respectively in the 2nd, 4 weeks; B group (Tpp65 dna immunization group) every mice is injected 200 μ gpcDNA3.1-Tpp65 plasmid DNA in the 0th, 2,4 weeks through quadriceps femoris respectively; C group (Tpp65 protein and DNA combined immunization group) every mice is injected 200 μ gpcDNA3.1-Tpp65 plasmid DNA in the 0th, 2 weeks through quadriceps femoris respectively, around the through the back subcutaneous multi-point injection 50 μ l Tpp65 protein (1 μ g/ μ l)+50 μ l CFA; D group (empty plasmid negative control group) every mice is injected 200 μ gpcDNA3.1 empty plasmids in the 0th, 2,4 weeks through quadriceps femoris respectively; E group (blank group) every mice is injected 200 μ l normal saline in the 0th, 2,4 weeks through quadriceps femoris respectively.3d after the last immunity, serum is collected in blood sampling, detects the pp65 antibody horizontal of respectively organizing in the mice serum; Simultaneously, get and respectively organize mouse boosting cell, as antigenic stimulus, mtt assay is measured the specific killing cell rate of lymphocytic proliferation index of T and spleen with reorganization pp65 protein; The double-antibody sandwich elisa standard measure detects the content of IFN-γ in the splenocyte supernatant.

The result: Tpp65 protein, Tpp65DNA, Tpp65 protein fragments+Tpp65 DNA combined immunization, empty plasmid negative control group, blank group, serum antibody titer is respectively: A group 1: 678.81; B group 1: 67.32; C group 1: 240; D organizes and E organizes all＜and 1: 10.Proliferation index is respectively: A group 6.76 ± 1.82; B group 5.43 ± 1.56; C group 7.18 ± 1.95; D group 2.74 ± 0.95; The CTL kill rate is respectively: A group 71.00% ± 18.82%; B group 56.86% ± 16.62%; C group 73.58% ± 22.61%; D group 23.58% ± 4.28%.IFN-γ is respectively: A organizes 747.66 ± 46.17ng/L; B organizes 464.12 ± 29.95ng/L; C organizes 787.97 ± 56.90ng/L; D organizes 100.06 ± 9.76ng/L

But pp65 protein vaccine distinguishing feature just is not only inducing cell immunity but also can induces humoral immunization, though at the inducing cell immunology inferior to Tpp65 protein and DNA combined immunization group, but the pp65 protein vaccine but can induce body to produce very high tire and antibody that specificity is very strong, and it is with strong points, side effect is little, and is safe.This vaccine is mainly for prevention parent HCMV primary infection and blocking-up HCMV congenital infection.

The specific embodiment

1, the extraction of genes of interest, amplification, purification

1.1 in HCMV AD169 type strain virus inoculation the pure man embryo fibroblast, when the characteristic pathological changes appears in the cell of propagation to 90%, collect smudge cells, the centrifuging and taking supernatant, adding E.C. 3.4.21.64 to final concentration is 500ng/L, 37 ℃ act on 1 hour, add 10%SDS to final concentration be 1%, 37 ℃ act on 1 hour, phenol: chloroform: isoamyl alcohol (25: 24: 1) extracting twice, chloroform: isoamyl alcohol (24: 1) extracting once adds extract in the bag filter, with distilled water dialysis 48 hours, change liquid once in every 6-8 hour, and concentrated 4 ℃ of preservations with PEG (molecular weight 20000).

1.2 utilize round pcr, be template amplification pp65 (1006-1521nt) gene (being called for short pp65) with HCMVDNA

1.3PCR the purification of product is operated by the commercial kit description, and the PCR product behind the purification is placed 4 ℃ of preservations.

2, being connected of pp65 genes of interest and prokaryotic expression carrier TOPO, conversion, evaluation

2.1 set up following reactant mixture system:

Pp65DNA:2 μ l through PCR test kit purification

Saline solution (TOPO support agent box is worn): 1 μ l

Aseptic double-distilled water: 2 μ l

TOPO carrier: 1 μ l

2.2 with above-mentioned reactant mixture uniform mixing, at room temperature (22 ℃-25 ℃) hatch 5min.

2.3 the junctional complex (mixture that contains junctional complex) of 3 μ l is added a pipe TOP10 competent cell, place 5min, 42 ℃ of 90sec on ice, place 1-2min immediately on ice, (SOC is that a kind of its composition of generally acknowledged culture medium has tryptone to add 800 μ lSOC, glucose, yeast extract, deionized water, NaCl), 37 ℃ are slowly shaken bacterium 1h, coating LB flat board (containing 100 μ g/ μ lAmp).Room temperature is placed 15min, is inverted overnight incubation for 37 ℃.

2.4 the plasmid of positive colony extracts

2.5PCR identify: recombiant plasmid being placed boiling water bath 5min, place immediately on ice, is that template and pp65 primer carry out the PCR reaction with it respectively, does 1.5% agarose gel electrophoresis analysis then.Primer:

p1：5’-CACCATGGATATCGACTTGCTGCTGC3’，

p2：5’-TCAATTCTGACCCTGAACCGTAGCCACC-3’

Giving birth to worker bio-engineering corporation by Shanghai provides.

2.6 the order-checking of positive colony is identified: carry out dna sequencing, sequencing result is seen accompanying drawing.

3, the conversion of recombination, expression and evaluation

3.1 the recombinant plasmid transformed E.coli BL21 host bacterium of process evaluation, purification,

3.2 pp65 recombiant protein abduction delivering:,

3.2.1 select positive bacteria drop point kind in 3ml Amp/LB (in the fluid medium of ampicillin/LB), 37 ℃ of shaken cultivation spend the night,

3.2.2 get 1: 50 amplification culture, 37 ℃ of vibration 4h next day, treat optical density absorption value OD ₆₀₀(referring to 600 nanometers optical density absorption values) value reaches at 0.6 o'clock, leaves and takes bacterium liquid 1ml, and 4000r/min abandons supernatant, for not inducing pipe.

3.2.3 adding 1ml IPTG (referring to isopropylthio-) in the bacterium liquid of remainder is 1mmol/L to final concentration, 37 ℃ of vibrations, leaves and takes bacterium liquid 1ml respectively after inducing 6h, the centrifugal 5min of 4000rpm stays bacterial sediment,

3.2.4 add albumen sample-loading buffer (composition: 100mmol/lTris-cl PH6.8,200mmol/l dithiothreitol, DTT, 4%SDS (electrophoresis level), 0.2% bromophenol blue, 20% glycerol.) 100 μ l, put 8min cracking antibacterial in the boiling water ,-20 ℃ are frozen standby.

3.3 expression of recombinant proteins product S DS-PAGE protein electrophoresis: 5% concentrates glue, 80v and 12% separation gel, 150v.Stripping glue, dyeing, decolouring.

3.4 the Western-blotting method detects the immunogenicity of marking protein, the results are shown in accompanying drawing.

4, a large amount of abduction deliverings of pp65 recombiant protein

4.1 strain is preserved:

The bacterial strain that the destination protein expression is higher is inoculated in the LB culture medium in the ratio of 1: 10 (w/v), and 37 ℃, 220rpm shaking table shaken cultivation are to OD ₆₀₀Be about 0.4-0.6, adding final concentration is the glycerol of 30%mg/ml, is distributed into the tubule of 1ml behind the mixing, and-80 ℃ of preservations are as original strain.

4.2 the abduction delivering of pp65 recombiant protein:

4.2.1-80 ℃ of frozen positive bacteria liquid of having identified are inoculated in the LB agar plate, 37 ℃ of overnight incubation.Next day, the single positive bacterium colony of picking from the flat board.

4.2.2 picking list colony inoculation is in 37 ℃ of 3ml LB culture medium, 220rpm shaking table shaken cultivation.

4.2.3 be inoculated in 37 ℃ of 50ml LB culture medium with 1: 100 ratio once more, 220rpm shaking table shaken cultivation is to OD ₆₀₀Be about 0.6-0.8.

4.2.4 be inoculated in 37 ℃ of 300ml LB culture medium with 1: 100 ratio once more, 220rpm shaking table shaken cultivation is to OD ₆₀₀Be about 0.4-0.6.

4.2.5 adding IPTG (derivant isopropylthio-) is 1mmol/L to final concentration, 5h is cultivated in continuation.

4.2.6 with the centrifugal 10min of 4000rpm, collect thalline,

4.2.7 (composition: every 1000ml contains NaCl 8.0g, KH with PBS ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, KCl 0.2g) behind twice of the buffer washing thalline, take by weighing the thalline weight in wet base, in-20 ℃ of preservations.

4.3 proteinic preparation:

4.3.1 the fragmentation of great expression thalline: adopt sonioation method ultrasonication thalline, extract the thalline suspension at 4 ℃ of centrifugal 10min of 12000rpm, abandon supernatant, precipitation is resuspended with ultrasonication liquid (referring to 20mmol/LTris-clPH8.0 100mmol/l NaCl 2mmol/LEDTA), continue to repeat above step 3-4 time, be after centrifugal to supernatant limpid till.

4.3.2 the washing of inclusion body and purification: the thalline behind the ultrasonic degradation is abandoned supernatant and every part of precipitation is resuspended in TE (the 20mmol/LTris-cl PH8.0 that 1ml contains 2M carbamide at 4 ℃ of centrifugal 10min of 12000rpm, 2mmol/LEDTA) in the buffer, continue to repeat above step 4-5 time, to going out supernatant precipitation is resuspended in the 1ml water, sucking-off 10 μ l samples from precipitation, mix with the 2XSDS-PAGE gel loading buffer, analyze, to determine protein washing degree by the SDS-PAGE electrophoresis.

4.3.4 quantification of protein: utilize ultraviolet spectrophotometer method.

Claims

1, the preparation method of human macrocell virus pp 65 protein vaccine, it is characterized in that extracting HCMV DNA, and be pp65 (1006-1521nt) genetic fragment that template amplification goes out to have CTL and bone-marrow-derived lymphocyte antigen advantage epi-position with it, be cloned into the PET100-TOPO prokaryotic expression carrier, and in E.coli BL21 cell, carry out the expression of external source genes of interest, identify its specificity and immunogenicity, a large amount of extraction and purification pp65 purpose fragment protein.