CN108300730A - 含有人巨细胞病毒ul140基因的重组质粒、基因工程菌及其应用 - Google Patents
含有人巨细胞病毒ul140基因的重组质粒、基因工程菌及其应用 Download PDFInfo
- Publication number
- CN108300730A CN108300730A CN201810108126.9A CN201810108126A CN108300730A CN 108300730 A CN108300730 A CN 108300730A CN 201810108126 A CN201810108126 A CN 201810108126A CN 108300730 A CN108300730 A CN 108300730A
- Authority
- CN
- China
- Prior art keywords
- human cytomegalovirus
- containing human
- genes
- genetic engineering
- engineering bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000701024 Human betaherpesvirus 5 Species 0.000 title claims abstract description 52
- 239000013612 plasmid Substances 0.000 title claims abstract description 48
- 241000894006 Bacteria Species 0.000 title claims abstract description 42
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 22
- 230000014509 gene expression Effects 0.000 claims abstract description 20
- 238000005215 recombination Methods 0.000 claims description 23
- 230000006798 recombination Effects 0.000 claims description 23
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 19
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 19
- 101150114134 UL140 gene Proteins 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 241000700605 Viruses Species 0.000 claims description 7
- 239000000969 carrier Substances 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 230000002068 genetic effect Effects 0.000 claims description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 5
- 241000701022 Cytomegalovirus Species 0.000 claims description 3
- 241001597008 Nomeidae Species 0.000 claims description 3
- 230000001413 cellular effect Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000001042 affinity chromatography Methods 0.000 claims description 2
- 102000037865 fusion proteins Human genes 0.000 abstract description 14
- 108020001507 fusion proteins Proteins 0.000 abstract description 14
- 238000000746 purification Methods 0.000 abstract description 8
- 239000000523 sample Substances 0.000 abstract description 7
- 210000000987 immune system Anatomy 0.000 abstract description 5
- 208000032420 Latent Infection Diseases 0.000 abstract description 4
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 abstract description 4
- 230000007246 mechanism Effects 0.000 abstract description 4
- 239000013604 expression vector Substances 0.000 abstract description 3
- 102000019034 Chemokines Human genes 0.000 abstract 1
- 108010012236 Chemokines Proteins 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 27
- 239000000243 solution Substances 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 18
- 239000000047 product Substances 0.000 description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 16
- 239000000741 silica gel Substances 0.000 description 16
- 229910002027 silica gel Inorganic materials 0.000 description 16
- 229960001866 silicon dioxide Drugs 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 14
- 239000000499 gel Substances 0.000 description 13
- 230000029087 digestion Effects 0.000 description 12
- 239000000427 antigen Substances 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 238000002156 mixing Methods 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 8
- 239000003292 glue Substances 0.000 description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 6
- 108010024164 HLA-G Antigens Proteins 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 6
- 150000002460 imidazoles Chemical class 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000002699 waste material Substances 0.000 description 6
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000002147 killing effect Effects 0.000 description 5
- 238000004064 recycling Methods 0.000 description 5
- 230000009182 swimming Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 230000000890 antigenic effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 210000000822 natural killer cell Anatomy 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 239000012474 protein marker Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 230000035939 shock Effects 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 101000984190 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 1 Proteins 0.000 description 2
- 101000984189 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 2 Proteins 0.000 description 2
- 102100025584 Leukocyte immunoglobulin-like receptor subfamily B member 1 Human genes 0.000 description 2
- 102100025583 Leukocyte immunoglobulin-like receptor subfamily B member 2 Human genes 0.000 description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 101710090398 Viral interleukin-10 homolog Proteins 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 108010068380 arginylarginine Proteins 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 239000006167 equilibration buffer Substances 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- SFNFGFDRYJKZKN-XQXXSGGOSA-N Ala-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C)N)O SFNFGFDRYJKZKN-XQXXSGGOSA-N 0.000 description 1
- YHKANGMVQWRMAP-DCAQKATOSA-N Ala-Leu-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YHKANGMVQWRMAP-DCAQKATOSA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 1
- IEAUDUOCWNPZBR-LKTVYLICSA-N Ala-Trp-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N IEAUDUOCWNPZBR-LKTVYLICSA-N 0.000 description 1
- DQNLFLGFZAUIOW-FXQIFTODSA-N Arg-Cys-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O DQNLFLGFZAUIOW-FXQIFTODSA-N 0.000 description 1
- FKQITMVNILRUCQ-IHRRRGAJSA-N Arg-Phe-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O FKQITMVNILRUCQ-IHRRRGAJSA-N 0.000 description 1
- VRTWYUYCJGNFES-CIUDSAMLSA-N Arg-Ser-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O VRTWYUYCJGNFES-CIUDSAMLSA-N 0.000 description 1
- IARGXWMWRFOQPG-GCJQMDKQSA-N Asn-Ala-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IARGXWMWRFOQPG-GCJQMDKQSA-N 0.000 description 1
- KSBHCUSPLWRVEK-ZLUOBGJFSA-N Asn-Asn-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O KSBHCUSPLWRVEK-ZLUOBGJFSA-N 0.000 description 1
- KGAJCJXBEWLQDZ-UBHSHLNASA-N Asp-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N KGAJCJXBEWLQDZ-UBHSHLNASA-N 0.000 description 1
- YDJVIBMKAMQPPP-LAEOZQHASA-N Asp-Glu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O YDJVIBMKAMQPPP-LAEOZQHASA-N 0.000 description 1
- OMMIEVATLAGRCK-BYPYZUCNSA-N Asp-Gly-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)NCC(O)=O OMMIEVATLAGRCK-BYPYZUCNSA-N 0.000 description 1
- ZVGRHIRJLWBWGJ-ACZMJKKPSA-N Asp-Ser-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVGRHIRJLWBWGJ-ACZMJKKPSA-N 0.000 description 1
- OZBXOELNJBSJOA-UBHSHLNASA-N Asp-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N OZBXOELNJBSJOA-UBHSHLNASA-N 0.000 description 1
- JSHWXQIZOCVWIA-ZKWXMUAHSA-N Asp-Ser-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JSHWXQIZOCVWIA-ZKWXMUAHSA-N 0.000 description 1
- 108010073254 Colicins Proteins 0.000 description 1
- GGIHYKLJUIZYGH-ZLUOBGJFSA-N Cys-Cys-Asp Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N)C(=O)O GGIHYKLJUIZYGH-ZLUOBGJFSA-N 0.000 description 1
- 230000012215 ER to Golgi vesicle-mediated transport Effects 0.000 description 1
- VUVKKXPCKILIBD-AVGNSLFASA-N Gln-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N VUVKKXPCKILIBD-AVGNSLFASA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- NHMRJKKAVMENKJ-WDCWCFNPSA-N Gln-Thr-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NHMRJKKAVMENKJ-WDCWCFNPSA-N 0.000 description 1
- WIKMTDVSCUJIPJ-CIUDSAMLSA-N Glu-Ser-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WIKMTDVSCUJIPJ-CIUDSAMLSA-N 0.000 description 1
- IBYOLNARKHMLBG-WHOFXGATSA-N Gly-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IBYOLNARKHMLBG-WHOFXGATSA-N 0.000 description 1
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 1
- 102100028976 HLA class I histocompatibility antigen, B alpha chain Human genes 0.000 description 1
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 description 1
- 108010075704 HLA-A Antigens Proteins 0.000 description 1
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 1
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 1
- 108010058607 HLA-B Antigens Proteins 0.000 description 1
- XIGFLVCAVQQGNS-IHRRRGAJSA-N His-Pro-His Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 XIGFLVCAVQQGNS-IHRRRGAJSA-N 0.000 description 1
- DGLAHESNTJWGDO-SRVKXCTJSA-N His-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N DGLAHESNTJWGDO-SRVKXCTJSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 description 1
- LPXHYGGZJOCAFR-MNXVOIDGSA-N Ile-Glu-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N LPXHYGGZJOCAFR-MNXVOIDGSA-N 0.000 description 1
- KBDIBHQICWDGDL-PPCPHDFISA-N Ile-Thr-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N KBDIBHQICWDGDL-PPCPHDFISA-N 0.000 description 1
- HQLSBZFLOUHQJK-STECZYCISA-N Ile-Tyr-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N HQLSBZFLOUHQJK-STECZYCISA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 1
- WXDRGWBQZIMJDE-ULQDDVLXSA-N Leu-Phe-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(O)=O WXDRGWBQZIMJDE-ULQDDVLXSA-N 0.000 description 1
- MJWVXZABPOKJJF-ACRUOGEOSA-N Leu-Phe-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MJWVXZABPOKJJF-ACRUOGEOSA-N 0.000 description 1
- PWPBLZXWFXJFHE-RHYQMDGZSA-N Leu-Pro-Thr Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O PWPBLZXWFXJFHE-RHYQMDGZSA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 1
- RDFIVFHPOSOXMW-ACRUOGEOSA-N Leu-Tyr-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RDFIVFHPOSOXMW-ACRUOGEOSA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical class [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- NDJSSFWDYDUQID-YTWAJWBKSA-N Met-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N)O NDJSSFWDYDUQID-YTWAJWBKSA-N 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- 241001416149 Ovis ammon Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- WMGVYPPIMZPWPN-SRVKXCTJSA-N Phe-Asp-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N WMGVYPPIMZPWPN-SRVKXCTJSA-N 0.000 description 1
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 1
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- RFWXYTJSVDUBBZ-DCAQKATOSA-N Pro-Pro-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RFWXYTJSVDUBBZ-DCAQKATOSA-N 0.000 description 1
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 101000999689 Saimiriine herpesvirus 2 (strain 11) Transcriptional regulator ICP22 homolog Proteins 0.000 description 1
- MESDJCNHLZBMEP-ZLUOBGJFSA-N Ser-Asp-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MESDJCNHLZBMEP-ZLUOBGJFSA-N 0.000 description 1
- FTVRVZNYIYWJGB-ACZMJKKPSA-N Ser-Asp-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O FTVRVZNYIYWJGB-ACZMJKKPSA-N 0.000 description 1
- OLKICIBQRVSQMA-SRVKXCTJSA-N Ser-Ser-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O OLKICIBQRVSQMA-SRVKXCTJSA-N 0.000 description 1
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- NJEMRSFGDNECGF-GCJQMDKQSA-N Thr-Ala-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O NJEMRSFGDNECGF-GCJQMDKQSA-N 0.000 description 1
- OJRNZRROAIAHDL-LKXGYXEUSA-N Thr-Asn-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O OJRNZRROAIAHDL-LKXGYXEUSA-N 0.000 description 1
- VGYBYGQXZJDZJU-XQXXSGGOSA-N Thr-Glu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VGYBYGQXZJDZJU-XQXXSGGOSA-N 0.000 description 1
- FHDLKMFZKRUQCE-HJGDQZAQSA-N Thr-Glu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FHDLKMFZKRUQCE-HJGDQZAQSA-N 0.000 description 1
- BDGBHYCAZJPLHX-HJGDQZAQSA-N Thr-Lys-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BDGBHYCAZJPLHX-HJGDQZAQSA-N 0.000 description 1
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- OCCYDHCUKXRPSJ-SXNHZJKMSA-N Trp-Ile-Gln Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O OCCYDHCUKXRPSJ-SXNHZJKMSA-N 0.000 description 1
- NOXKHHXSHQFSGJ-FQPOAREZSA-N Tyr-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NOXKHHXSHQFSGJ-FQPOAREZSA-N 0.000 description 1
- 101710153113 Unique short US2 glycoprotein Proteins 0.000 description 1
- 101710126011 Unique short US6 glycoprotein Proteins 0.000 description 1
- VNGKMNPAENRGDC-JYJNAYRXSA-N Val-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=CC=C1 VNGKMNPAENRGDC-JYJNAYRXSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000021040 cytoplasmic transport Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 210000000224 granular leucocyte Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 108091008042 inhibitory receptors Proteins 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 108010060175 trypsinogen activation peptide Proteins 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/088—Varicella-zoster virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
- C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种含有人巨细胞病毒UL140基因的重组质粒、基因工程菌及其应用。该含有人巨细胞病毒UL140基因的重组质粒是通过将UL140基因插入表达型载体pET32a中获得;然后将其转进表达菌BL21中,构建能表达聚组氨酸标签HCMV UL140融合蛋白的工程菌株,并利用该工程菌表达获得对应的HCMV UL140融合蛋白。本发明中获得的融合蛋白可应用亲和层析纯化后制备多克隆抗体,从而有利于探究趋化因子蛋白HCMV UL140如何规避宿主免疫系统的逃避机制,降低HCMV潜伏感染宿主细胞的数量提高机体的免疫识别。
Description
技术领域
本发明涉及生物学领域,特别涉及一种含有人巨细胞病毒UL140基因的重组质粒、基因工程菌及其应用。
背景技术
巨细胞病毒感染是由人巨细胞病毒(HCMV)引起的常见病毒性感染之一。在新生儿、免疫低下和免疫抑制病人(如艾滋病、器官移植或肿瘤)可引起严重感染,甚至导致死亡。HCMV感染的发病情况与机体的免疫功能状态关系密切。原发性HCMV感染时,病毒在人体血液中以单核和多形核白细胞为载体,扩散至全身各个器官。
HCMV在正常人群中传播极为广泛,很少导致明显疾病,大多数人呈潜伏感染。但这对免疫减弱或损伤的人群(免疫缺陷、器官移植、肿瘤、艾滋病病人和孕妇)构成极大威胁。因此,亟待建立新的诊断方法的研究,提高对潜伏感染、少量持续性感染和侵袭性HCMV感染的检测能力。细胞毒性T细胞(CTL)是肿瘤多肽疫苗诱导机体产生抗肿瘤反应的主要细胞,抗原诱导CTL反应经过3个基本反应:蛋白酶体降解抗原蛋白为多肽片段、抗原加工相关转运体将多肽片段转运至内质网、MHC-Ⅰ类分子与抗原肽结合并提呈。人类白细胞抗原是人的主要组织相容性复合物,在移植排斥反应中起关键作用,降低HLA的表达,可以延长移植物的存活时间(W.E保罗,Fundamental Immunology,2003)。
HCMV可通过下调经典MHC-Ⅰ和MHC-Ⅱ类分子表达,抑制抗原提成细胞(APC)的抗原提呈功能和CD8+CTL对靶细胞的杀伤。MHC-Ⅰ类分子的下降,会增加被NK细胞识别和杀伤的几率,但HCMV感染细胞可表达非经典的MHC-Ⅰ类分子HLA-G、HLA-E、和MHC-Ⅰ类分子同源物,与NK细胞的杀伤抑制性受体结合,抑制NK细胞对感染细胞的杀伤,逃避被机体免疫系统攻击。HCMV实验株与临床株相比缺少了UL/b′UL128-UL150,这簇基因虽然不是病毒的必须基因,但是对于人巨细胞病毒逃避免疫和感染上皮细胞、EC细胞有作用。人巨细胞病毒干扰MHC-Ⅰ呈递抗原主要有两种途径:①干扰TAP肽转运②固定插入内质网膜的HLA重链。HCMVTB40/E-UL140蛋白BIMAS:HLAPeptide Binding Predictions与MHC-Ⅰ有非常高的亲和性。免疫细胞识别MHC-Ⅰ呈递的抗原后,CD8 +细胞毒性T细胞前体细胞与MHC-Ⅰ呈递抗原结合活化为成熟CTL,有助于机体清除病毒感染的细胞,防止病毒的进一步传播。
Hegde等研究发现HCMV gpUS3可在内质网中与MHC-Ⅱ类分子的α和β链结合,抑制其由内质网向高尔基体转运,使其在细胞表面表达下降。HCMVgpUS2可与HLA-DR-α和HLA-DM-α链结合并使其降解。HCMV gpUS2和gpUS3可以可溶性表达于细胞外,被未感染的APC细胞摄取,抑制未感染的APC细胞呈递抗原给CD4+T细胞。HLA-G是一种非经典的MHC-Ⅰ类分子,可分为HLA1-6六种受体,HLA-G的受体为白细胞免疫球蛋白受体1和2(LIR1和LIR2),为抑制性受体。LIR1表达于NK、T、B和粒细胞表面,LIR2主要表达于单核细胞、巨噬细胞和树突状细胞表面。HLA-G与LIR结合,可抑制抗原提呈细胞的抗原提呈功能,可抑制HLA-A2限制性的人巨细胞病毒诱导的CTL杀伤活性,可融性HLA-G可抑制NK细胞对K562细胞株的杀伤活性,HCMV可通过诱导受感染细胞表达HLA-G而逃避免疫应答。
pET载体最初由Studier及其同事构建。Novagen开发的系列新的pET载体则使目的蛋白的克隆、检测以及纯化更加容易。pET32系列载体是设计用来克隆构建和高水平融合表达表达带有109个氨基酸Trx标签的蛋白质序列。将基因插入到载体的多克隆位点,能够获得融合表达蛋白质。
HCMV在与人类共进化的过程中,形成了多种分子机制逃避机体免疫系统的监视,从而在体内长期潜伏。GpUS3是HCMV表达的一种即刻早期蛋白,可在内质网与HLA-A,HLA-B分子的α重链结合,阻碍MHC-Ⅰ分子装配成熟及从内质网中转运至细胞膜表面(Bullock GC,et al.2001)。HCMV gpUS6在胞浆中可与TAP结合,阻止抗原肽从胞浆转运至内质网,使MHC-Ⅰ分子不能结合抗原肽,不能在细胞表面表达MHC-Ⅰ分子和抗原肽复合体。HCMV可编码一种白细胞介素-10类似物cmvIL-10,cmvIL-10可降低IFN-γ的表达,导致MHC-Ⅱ表达的分子降低。上述相关工作都为后续研究HCMV免疫逃逸提供了丰富的理论依据(Juliet V,etal.2002)。
构建含有UL140重组质粒的基因工程菌的发明将有助于研究UL140基因在人巨细胞病毒免疫逃避中的分子机制。一旦该分子机制被解锁,UL140基因可能会用作人巨细胞病毒感染的治疗靶点,可以为人类社会做出巨大贡献。但目前尚未有构建成功的含有UL140重组质粒的基因工程菌。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种含有人巨细胞病毒UL140基因的重组质粒。
本发明的另一目的在于提供含有上述人巨细胞病毒UL140基因重组质粒的基因工程菌。
本发明的又一目的在于提供上述含有巨细胞病毒UL140基因重组质粒的基因工程菌的应用。
本发明的目的通过下述技术方案实现:一种含有人巨细胞病毒UL140基因的重组质粒,含有UL140基因片段,其中,UL140基因片段的核苷酸序列如SEQID No:1所示。
所述的含有人巨细胞病毒UL140基因的重组质粒的构建方法,通过常规方法将UL140基因片段插入到pET32a载体中获得。
一种含有人巨细胞病毒UL140基因重组质粒的基因工程菌,转化有上述含有人巨细胞病毒UL140基因的重组质粒。
所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌的构建方法,为将上述含有人巨细胞病毒UL140基因的重组质粒转化入原核表达工程菌中,获得含有人巨细胞病毒UL140基因重组质粒的基因工程菌。
所述的基因工程菌优选为大肠杆菌;更优选为大肠杆菌BL21(DE3)。
所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌在UL140重组蛋白制备中的应用。
一种UL140重组蛋白的制备方法,将上述含有人巨细胞病毒UL140基因重组质粒的基因工程菌经过活化培养和发酵培养,再加入诱导剂IPTG进行诱导表达,得到UL140重组蛋白;具体为:将上述工程菌接种至含有氨苄青霉素(Amp)抗性的LB液体培养基中进行活化培养,然后转接到含有氨苄青霉素抗性LB液体培养基中进行发酵培养,再加入诱导剂IPTG进行诱导表达,得到UL140重组蛋白。
所述的氨苄青霉素的终浓度为100μg/ml。
所述的诱导表达的条件优选为:20℃诱导8h。
所述的IPTG的工作浓度为1mM/L。
所述的UL140重组蛋白的制备方法,还包括将获得的UL140重组蛋白进行纯化的步骤。
所述的纯化为采用亲和层析进行纯化。
所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌在制备人巨细胞病毒UL140蛋白多克隆抗体中的应用;该工程菌可以表达出带有组氨酸标签的融合蛋白,可应用亲和层析纯化制备多克隆抗体。
本发明相对于现有技术具有如下的优点及效果:
1、本发明提供了一种含有pET32a-UL140重组质粒的基因工程菌,通过将UL140基因插入表达型载体pET32a中获得重组载体pET32a-UL140,之后将其热击转进表达菌BL21中构建了能表达聚组氨酸标签HCMV UL140融合蛋白的工程菌株,菌株可表达获得对应的HCMVUL140融合蛋白,该融合蛋白可应用亲和层析纯化制备多克隆抗体,从而有利于探究趋化因子蛋白HCMV UL140如何规避宿主免疫系统的逃避机制,降低HCMV潜伏感染宿主细胞的数量提高机体的免疫识别。
2、在本发明构建的pET32a-UL140重组质粒中,基因的表达是受T7噬菌体RNA聚合酶调控的。载体的典型特点是带有pBR322的大肠菌素E1复制区,从而赋予宿主菌氨苄青霉素抗性。编码序列在多克隆位点插入,在T7lac启动子的控制之下,聚组氨酸标签的目的蛋白大量诱导表达,便于后续亲和纯化目的蛋白利于制作多克隆抗体,分析免疫逃逸机制。鉴于pET32a载体融合蛋白表达纯化的特性,已大量应用在病原微生物等抗原的大量制备,为探究UL140是如何逃避宿主免疫系统提供了理论基础。
附图说明
图1为pET32a-UL140重组质粒构造示意图。
图2为PCR扩增产物电泳示意图;其中,泳道M为DNA marker;泳道1为UL140PCR扩增产物。
图3为重组融合蛋白UL140(未纯化)的SDS-PAGE结果示意图;其中,泳道M为蛋白质Marker;箭头所示为重组融合蛋白UL140(大小为40KD)。
图4为重组蛋白的Western blot结果图;其中,泳道M为预染蛋白质Marker;箭头所示为重组融合蛋白UL140(大小为40KD)。
图5为重组融合蛋白UL140纯化的SDS-PAGE结果示意图;其中,泳道M为蛋白质Marker;箭头所示为重组融合蛋白UL140(大小为40KD)。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。
一、质粒构建
(1)UL140基因引物设计
根据GennBank上公布的TB40/E(KF297339.1)病毒株基因组中UL140基因的序列全长576bp(核苷酸序列如SEQ ID No:1所示,蛋白翻译产物如SEQID No:2所示),以该序列为模板,以pET32a载体(优宝生物有限公司)为克隆载体和表达载体,使用DNAstar软件,选取合适的酶切位点进行引物设计。设计好的引物如下表:
表1UL140引物设计表
注:F(上游引物);R(下游引物)。
(2)UL140基因PCR
根据已发表在GenBank上的HCMV TB40/E-BAC的基因序列(GenBank:EF999921.1,引用文章(Cloning and sequencing of a highly productice,endotheliotropic virusstrain derived from human cytomegalovirus TB40/E))为模板,在以下反应体系中进行PCR:
表2PCR反应体系表
试剂名称 | 体积(25μl) |
无菌ddH2O | 14.9μl |
5×PCR buffer(with Mg2+) | 5μl |
dNTP | 2μl |
模板 | 1μl |
上游引物 | 1μl |
下游引物 | 1μl |
Fast pfu酶 | 0.1μl |
PCR扩增条件见下表:
表3PCR反应条件设置表
PCR产物经1%的琼脂糖凝胶分析,并通过胶回收试剂盒回收PCR产物。PCR产物回收步骤如下:
①PCR产物经电泳后,在紫外条件下切取含目的条带的凝胶置于干净的1.5mL的EP管中;
②在EP管中加入与凝胶等体积的XP2溶液;
④将混有XP2溶液的凝胶块的1.5mL的EP管置于水浴锅中,57℃加热,每隔2~3min颠倒混匀,直至凝胶块完全融化即可;
③待完全融化后的凝胶冷却,便可将其斡旋混匀并短暂离心,最后将其加入事先组装好的硅胶柱,室温,10000×g,离心1min,倾倒收集管中的废液;
⑤再加入300μL XP2溶液到硅胶柱中,室温10000×g离心1min后,将收集管中的液体倒掉;
⑥用700μL SPW洗涤溶液洗涤硅胶柱,室温10000×g离心1min,将收集管中的液体倒掉;
⑦重复6步骤一次;
⑧空甩硅胶柱,室温12000×g离心2min,去除多余的SPW洗涤溶液;
⑨取出硅胶柱并将其放入到灭菌的1.5mL EP管中,室温放置2min后,向硅胶柱中加入30μL事先已加热到65℃无菌ddH20,室温静置1min后,12000×g离心1min,取出已废弃的硅胶柱,EP管中手记的液体即为目的DNA溶液;
⑩检测回收的目的DNA浓度及纯度,样品放于-20℃保存。
实验结果:PCR扩增产物如图2所示,与预期大小结果一致,目的基因片段大小为576bp。
(3)产物纯化试剂盒回收PCR产物
①将PCR扩增后产物转移到一个干净的1.5mL EP管,根据PCR扩增体系加入4~5倍的结合液CP,充分混匀;
②将上一步所得溶液加入吸附柱EC中(吸附柱放入收集管中),室温放置1min,10000×g,离心1min,倾倒收集管中的废液;
③加入700μl漂洗液WB(先检查是否已加入无水乙醇),10,000g离心1min,弃掉废液;
④重复3步骤一次;
⑤将吸附柱EC放回空收集管中,10000×g,离心1min,尽可能去除漂洗液,漂洗液中残留乙醇会抑制下游反应;
⑥取出吸附柱EC,放入一个干净的离心管中,在吸附膜的中间部位加30~50μl洗脱缓冲液EB(洗脱缓冲液事先在65~70℃水浴中加热效果更好),室温放置2min,13000×g离心1min。如果需要较多量DNA,可将得到的溶液重新加入吸附柱中,离心1min。取出已废弃的硅胶柱,EP管中手记的液体即为目的DNA溶液;
⑦检测回收的目的DNA浓度及纯度,以更好的用于后续实验。样品放于-20℃保存。
(4)pET32a质粒抽提
①取出过夜培养好的含有pET32a质粒的DH5α菌液,并将菌体加到1.5mLEP管中,12000×g室温离心1min,弃上清,重复步骤,将5mL菌液全部收集到1.5mL EP管中;
②吸净多余的上清后,加250μl solution I到EP管中,用移液器轻轻吹打菌体沉淀,待菌体重悬后,放于涡旋振器上,使其完全混匀;
③再加入250μl solution II,轻轻地上下颠倒混匀数次,颠倒时间要把握好,控制在2min~5min之间,颠倒完之后,液体一般会透明澄清;
④最后加入350μl solution III,轻轻地上下颠倒混匀数次,至有白色沉淀形成,颠倒混匀次数不能过多,紧接着放于小型离心机中,室温,13000×g离心10min;
⑤离心结束后,将移液枪把上清液转移到已标记好的硅胶柱中,硅胶柱放进2ml的离心管里,勿将白色沉淀物吸入硅胶柱中,放于小型离心机中,室温10000×g,离心1min;
⑥倒掉收集管中的废液,吸取500μl HB溶液,加入到硅胶柱中,室温10000×g离心1min;
⑦倒掉收集管中的废液,加700μl DNA Wash Buffer到硅胶柱中,(先检查DNAWash Buffer是否已加入无水乙醇)室温10000×g离心1min;重复8步骤一次;
⑧室温13000×g离心2min,去除多余的DNA Wash Buffer;
⑨取出硅胶柱将其放入到灭菌的1.5mL EP管中,向硅胶柱中加入30μl~50μl事先已加热到65℃无菌ddH20,室温静置1~2min后,12000×g离心1min,于-20℃保存。
(5)载体和目的基因的酶切及回收
①pET32a载体和UL140基因PCR产物双酶切体系如下表:
表4PCR产物双酶切体系表
将抽提好的质粒与回收的PCR产物于37℃恒温水浴14min进行双酶切。
②pET32a双酶切后需要胶回收,同步骤(2)。
③目的UL140基因双酶切回收,具体步骤如下:
Ⅰ、根据双酶切体系加入4~5倍的结合液CP,充分混匀;
Ⅱ、将上一步所得溶液加入吸附柱EC中,室温放置1min,10000×g,离心1min,倒掉收集管中的废液;
Ⅲ、加入700μl漂洗液WB(先检查是否已加入无水乙醇),10000×g,离心1min,弃掉废液;
Ⅳ、重复3步骤一次;
Ⅴ、将吸附柱EC放回收集管中,10000×g,离心1min,尽可能除去漂洗液,以免残留乙醇抑制下游反应;
Ⅵ、取出吸附柱EC,放入一个干净的离心管中,在吸附膜的中间部位加30~50μl洗脱缓冲液EB(洗脱缓冲液事先在65~70℃水浴中加热效果更好);室温放置2min,13000×g离心1min。如果需要较多量DNA,可将得到的溶液重新加入吸附柱中,离心1min。取出已废弃的硅胶柱,EP管中手记的液体即为目的DNA溶液;
Ⅶ、检测回收的目的DNA浓度及纯度,以更好的用于后续实验。样品放于-20℃保存。
(6)UL140双酶切回收产物与pET32a质粒双酶切回收产物连接反应
连接反应体系如下表:
表5连接反应体系表
连接反应条件为:置于16℃连接仪中连接16h以上;获得重组质粒pET32a-UL140。
(7)大肠杆菌BL21(DE3)感受态细胞的制备
①从-80℃冰箱取出大肠杆菌BL21(DE3),于无抗性的LB固体培养基上划线接种,37℃培养12h;
②在划线过夜后的LB平板上,挑取单菌落,接种于4mL LB液体培养基中37℃培养过夜;
③将培养得到产物次日按1%(v/v)的比例,接种于500mL LB液体培养基中,37℃培养;
④当培养物OD600达0.5~0.6时,将培养物置冰上10~15min,8000r/min离心10min收集菌体;
⑤用预冷的TB buffer轻轻地将沉淀悬浮,8000r/min离心30min,如此重复洗涤2次;
⑥用18.6ml预冷TB buffer和1.4ml DMSO的轻轻将沉淀悬浮,-80℃保存备用。
(8)重组质粒转化进大肠杆菌BL21(DE3)
①取重组质粒pET32a-UL140 1μl加入BL21(DE3)感受态细胞中,冰上静置30min;
②将混有质粒的BL21(DE3)感受态细胞放入恒温水浴锅中42℃90S热击;
③热击后立即冰浴3min;
④冰浴后加入800μl SOB液体培养基(SOB培养基配方:2%(w/v)Tryptone、0.5%(w/v)Yeast Extract、0.05%(w/v)氯化钠、2.5mM氯化钾、10mM氯化镁),37℃培养1h,取出后将培养物8000r/min离心5min,弃去上清,只留下约100μl菌液,吹打混匀后将其涂于LB平板(含1%(v/v)100μg/ml Amp),放置37℃培养箱中倒置过夜培养12~24h即可看到单菌落。
(9)重组质粒鉴定
①无菌操作下,挑取单菌落,接种于10mL LB液体培养基(含1%(v/v)100μg/mlAmp)中,37℃培养8h后提取质粒,把1%琼脂糖核酸凝胶电泳鉴定为阳性的pET32a-UL140重组质粒作为模板,进行PCR鉴定。
表6PCR反应体系表
试剂名称 | 体积(20μl) |
无菌ddH2O | 14.9μl |
5×PCR buffer(with Mg2+) | 5μl |
dNTP | 2μl |
模板 | 1μl |
上游引物 | 1μl |
下游引物 | 1μl |
Fast pfu酶 | 0.1μl |
PCR扩增条件见下表:
表7PCR反应条件设置表
PCR扩增结束后,反应产物跑1%的琼脂糖凝胶电泳,凝胶成像仪拍照,并分析、保存实验结果。
②双酶切鉴定
(Ⅰ)抽提重组质粒pET32a-UL140同步骤(4);
(Ⅱ)重组质粒pET32a-UL140双酶切鉴定同步骤(5)。
(10)质粒样品测序
将可以双酶切出目的基因大小片段的质粒样品送公司测序,测序结果出来后,将测序结果与GennBank下载的UL140基因序列使用BLAST软件进行比对,分析比对结果。测序结果与下载序列匹配,可以进行下一步实验。
二、UL140重组蛋白的原核表达
(1)UL140重组蛋白的诱导表达鉴定
①重组载体pET32a-UL140诱导表达条件优化:pET32a-UL140表达最佳诱导条件是:20℃、1%(v/v)浓度为1mM的IPTG、8h。
②重组蛋白UL140的鉴定
Ⅰ.把鉴定好的pET32a-UL140重组BL21(DE3)菌按1%(v/v)接种于4mLLB液体培养基(含1%(v/v)100μg/ml Amp)中,37℃静止培养过夜。次日按1%(v/v)将活化的菌液接种于4mL LB液体培养基(含1%(v/v)100μg/mlAmp)中,37℃当菌液OD600约为0.5时加入1%(100mM)IPTG,20℃培养6h。8000r/min离心1min收集菌体,加入4×上样缓冲液(含DTT),用漩涡震荡器混匀,沸水浴中煮沸10min,l0000r/min离心30s,取15μl上清,以12%分离胶5mL,8%浓缩胶2mL进行配胶进行SDS聚丙烯酞胺凝胶电泳(SDS-PAGE)。
表8 12%分离胶配制表
试剂名称 | 体积(总5ml) |
无菌ddH2O | 1.6ml |
30%丙烯酰胺贮存胶 | 2.0ml |
分离胶缓冲液 | 1.3ml |
10%SDS | 50μl |
10%AP | 48μl |
TEMED | 2μl |
混匀后将试剂灌入玻璃板间,以水封顶,注意使液面水平,凝胶完全聚合需要30~60min;
配置5%浓缩胶见下表:
表9 5%浓缩胶配制表
试剂名称 | 体积(总5ml) |
无菌ddH2O | 3.4ml |
30%丙烯酰胺贮存胶 | 830μl |
浓缩胶缓冲液 | 630μl |
10%SDS | 50μl |
10%AP | 85μl |
TEMED | 5μl |
将分离胶上的水倒去,并用滤纸吸干多余水份,加入上述混合液,立即将梳子插入玻璃板间,完全聚合需15~30min;
Ⅱ.Western Blot步骤如下:
①首先将细胞蛋白经12%SDS-PAGE凝胶电泳进行分离并转移至PVDF膜上;
②用5%的脱脂奶粉室温封闭1h;
③封闭完的PVDF膜与anti-His鼠抗(1:1000)4℃共孵育过夜;
④用含有1/2000吐温的TBST洗3次,每次20min;
⑤洗后的膜与荧光二抗山羊抗鼠(1:5000)4℃共孵育2~3h;
⑥再用含有1/2000吐温的TBST洗3次,每次20min;
⑦最后通过LI-COR ODYSSEY FC成像系统进行曝光进行免疫印迹分析。
实验结果:未纯化的重组融合蛋白UL140的SDS-PAGE结果如图3所示。组蛋白的Western blot结果如图4所示。
三、UL140重组蛋白的纯化
(1)超声波破菌
①加入1L(1/20细胞生长体积)的1×PBS缓冲液重悬菌体,4℃,6,500×g,离心10min尽可能弃尽上清;
②加入的50ml 1×PBS缓冲液重悬菌体,采用超声波破碎仪进行破菌,破菌参数设置为:150W,工作15s,停5s,破碎30min~1h。
③破菌完成后,10000×g,4℃离心15min以上,收集上清液体1.5ml,用移液枪吸100μl沉淀,加到1.5ml EP管内,跑SDS-PAGE蛋白胶分析样品;其结果如图5所示,箭头所示为重组融合蛋白UL140,为40KD。
④剩下的沉淀加入30ml的平衡缓冲液,37℃震荡1h,冰上0.22μm滤膜过滤备用。
(2)UL140重组蛋白亲和层析纯化
①10倍柱体积的ddH2O(0.22μm)清洗Ni层析柱;
②10倍柱体积的平衡缓冲液(0.22μm)清洗Ni层析柱;
③平衡泵泵入重组蛋白溶液;
④分别用5倍柱体积5mM咪唑、50mM咪唑、100mM咪唑、150mM咪唑、200mM咪唑、500mM咪唑洗脱。
(3)UL140纯化重组蛋白的鉴定
UL140重组蛋白的亲和层析纯化鉴定同步骤二(1)②。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 暨南大学
<120> 含有人巨细胞病毒UL140基因的重组质粒、基因工程菌及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 576
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> UL140基因
<400> 1
tcacagggtc tgatgaagct gccaagagtc gtggctgtgg cgcagcgcgt tctgtacggc 60
gcgtttcacc gctttctgca tggccgctac cacgtcgggt gggagcggct ccggcggaag 120
ctcgatgagc agttgctgcg agtctcggcg ctcggtgtcc gccgtttcgt cggacgtggc 180
gtaaaaaacc gaggtggttg cccagtcgtc cacgctgtcg acggcctctg tcagtgccgg 240
gttgtcaaaa ccgccatcgg acgcgggtga taaaagaacg tacgatgaca cgctgttagt 300
acgactctcg tcgtcgctct gggaacgacg tgatggacga cggtagatga cctcgtcttg 360
ccacgcgtcg aagcggtcgc agcagcgctg gatccaagcg cagcgaagca gcttacggaa 420
cacgtcgttg ttccaaaagt agagcataaa gagaaagaaa agtagcgtaa taatgaagcc 480
gaaaacgacg agggtcggca gggcactacc gccgctgccg ttttttgtgt cgtgcgggtg 540
cacggtggta gtggcgttag tctgagctgg ggtcat 576
<210> 2
<211> 191
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> UL140蛋白
<400> 2
Met Thr Pro Ala Gln Thr Asn Ala Thr Thr Thr Val His Pro His Asp
1 5 10 15
Thr Lys Asn Gly Ser Gly Gly Ser Ala Leu Pro Thr Leu Val Val Phe
20 25 30
Gly Phe Ile Ile Thr Leu Leu Phe Phe Leu Phe Met Leu Tyr Phe Trp
35 40 45
Asn Asn Asp Val Phe Arg Lys Leu Leu Arg Cys Ala Trp Ile Gln Arg
50 55 60
Cys Cys Asp Arg Phe Asp Ala Trp Gln Asp Glu Val Ile Tyr Arg Arg
65 70 75 80
Pro Ser Arg Arg Ser Gln Ser Asp Asp Glu Ser Arg Thr Asn Ser Val
85 90 95
Ser Ser Tyr Val Leu Leu Ser Pro Ala Ser Asp Gly Gly Phe Asp Asn
100 105 110
Pro Ala Leu Thr Glu Ala Val Asp Ser Val Asp Asp Trp Ala Thr Thr
115 120 125
Ser Val Phe Tyr Ala Thr Ser Asp Glu Thr Ala Asp Thr Glu Arg Arg
130 135 140
Asp Ser Gln Gln Leu Leu Ile Glu Leu Pro Pro Glu Pro Leu Pro Pro
145 150 155 160
Asp Val Val Ala Ala Met Gln Lys Ala Val Lys Arg Ala Val Gln Asn
165 170 175
Ala Leu Arg His Ser His Asp Ser Trp Gln Leu His Gln Thr Leu
180 185 190
<210> 3
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> UL140引物-F
<400> 3
cgggaattca tgaccccagc tcagactaac g 31
<210> 4
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> UL140引物-R
<400> 4
gcgcggaagc ttctatcaca gggtctgatg aagctgc 37
Claims (9)
1.一种含有人巨细胞病毒UL140基因的重组质粒,其特征在于:含有UL140基因片段,其中,UL140基因片段的核苷酸序列如SEQ ID No:1所示。
2.根据权利要求1所述的含有人巨细胞病毒UL140基因的重组质粒,其特征在于:将UL140基因片段插入到pET32a载体中获得。
3.一种含有人巨细胞病毒UL140基因重组质粒的基因工程菌,其特征在于:转化有权利要求1中所述的含有人巨细胞病毒UL140基因的重组质粒。
4.根据权利要求3所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌,其特征在于:所述的基因工程菌为大肠杆菌。
5.权利要求1所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌在UL140重组蛋白制备中的应用。
6.一种UL140重组蛋白的制备方法,其特征在于:将权利要求3或4中所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌经过活化培养和发酵培养,再加入诱导剂IPTG进行诱导表达,得到UL140重组蛋白。
7.根据权利要求6所述的UL140重组蛋白的制备方法,其特征在于:
所述的诱导表达的条件为:20℃诱导8h。
8.根据权利要求6所述的UL140重组蛋白的制备方法,其特征在于:还包括将获得的UL140重组蛋白进行纯化的步骤;
所述的纯化为采用亲和层析进行纯化。
9.权利要求3或4所述的含有人巨细胞病毒UL140基因重组质粒的基因工程菌在制备人巨细胞病毒UL140蛋白多克隆抗体中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810108126.9A CN108300730A (zh) | 2018-02-02 | 2018-02-02 | 含有人巨细胞病毒ul140基因的重组质粒、基因工程菌及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810108126.9A CN108300730A (zh) | 2018-02-02 | 2018-02-02 | 含有人巨细胞病毒ul140基因的重组质粒、基因工程菌及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN108300730A true CN108300730A (zh) | 2018-07-20 |
Family
ID=62864079
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810108126.9A Pending CN108300730A (zh) | 2018-02-02 | 2018-02-02 | 含有人巨细胞病毒ul140基因的重组质粒、基因工程菌及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108300730A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007038316A2 (en) * | 2005-09-23 | 2007-04-05 | The Trustees Of Princeton University | Human cytomegalovirus latency promoting genes, related virus variants and methods of use |
CN106591496A (zh) * | 2017-01-19 | 2017-04-26 | 温州医科大学 | 一种涵盖全基因组的人巨细胞病毒基因pcr检测芯片 |
-
2018
- 2018-02-02 CN CN201810108126.9A patent/CN108300730A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007038316A2 (en) * | 2005-09-23 | 2007-04-05 | The Trustees Of Princeton University | Human cytomegalovirus latency promoting genes, related virus variants and methods of use |
CN106591496A (zh) * | 2017-01-19 | 2017-04-26 | 温州医科大学 | 一种涵盖全基因组的人巨细胞病毒基因pcr检测芯片 |
Non-Patent Citations (4)
Title |
---|
LINGLING HE ET AL.: "Preparation of polyclonal antibody against human cytomegalovirus UL146 gene product – α chemokine vCXC-1", 《INTERNATIONAL JOURNAL OF SCIENCE》 * |
TOMASEC,P. ET AL.: "Human herpesvirus 5 strain TB40/E clone Lisa, complete genome,GenBank: KF297339.1", 《GENBANK》 * |
柳志强: "《分子微生物学实验指导》", 31 July 2017, 中国轻工业出版社 * |
蔡丽君等: "广州地区人巨细胞病毒临床低传代株UL140 基因的多态性", 《热带医学杂志》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102827254B (zh) | 细胞穿膜肽hPP10及其用途 | |
CN113512096B (zh) | 一种鲈鱼弹状病毒重组g2蛋白及其应用 | |
CN103304637B (zh) | 细胞穿膜肽hPP3及其用途 | |
CN105106945B (zh) | 一种幽门螺旋杆菌四价毒力因子多表位疫苗及其制备方法 | |
CN103275223B (zh) | 降钙素原抗体的制备方法 | |
CN105126093B (zh) | 一种幽门螺旋杆菌四价黏附因子多表位疫苗及其制备方法 | |
CN108264548B (zh) | 鳜鱼伽玛干扰素相关因子及其重组蛋白和应用 | |
CN107596361B (zh) | 牛a型产气荚膜梭菌的亚单位疫苗及其制备方法和应用 | |
CN109111507A (zh) | 病毒重组糖蛋白及其真核细胞高效表达方法与应用 | |
CN102993278B (zh) | 耐甲氧西林金黄色葡萄球菌(MRSA)疫苗重组蛋白抗原FnbA1的纯化方法 | |
CN102898511B (zh) | 耐甲氧西林金黄色葡萄球菌重组基因工程疫苗候选抗原i12c制备中的纯化方法 | |
CN108359679A (zh) | 含有人巨细胞病毒ul147基因的重组质粒、基因工程菌及其应用 | |
CN105624082B (zh) | 表达单链抗体scFV或多肽适配体的功能化细菌磁颗粒及其应用 | |
CN104862331B (zh) | 一种可溶性表达马红球菌致病基因VapA蛋白的方法 | |
CN106754981A (zh) | 一种利用大肠杆菌系统制备小鹅瘟病毒样颗粒的方法 | |
Davies et al. | Definition of murine T helper cell determinants in the major capsid protein of human papillomavirus type 16 | |
CN108300730A (zh) | 含有人巨细胞病毒ul140基因的重组质粒、基因工程菌及其应用 | |
CN105420174A (zh) | 一种表达重组vegf融合蛋白的基因工程菌的构建 | |
CN112341525A (zh) | 一种重组非洲猪瘟病毒pE120R亚单位可溶性蛋白及其制备方法和应用 | |
CN104678097A (zh) | 一种用于肺结核诊断的结核分枝杆菌组合抗原 | |
CN113121672A (zh) | 猫干扰素γ的可溶性原核表达和纯化方法及应用 | |
CN113307852A (zh) | 一种用于恙虫病东方体47kDa蛋白的诱导表达及纯化方法 | |
CN113735943A (zh) | 一种重组的非洲猪瘟病毒p72亚单位蛋白和制备方法及其应用 | |
CN108588096B (zh) | 东方巴贝斯虫球状体蛋白基因4及其编码的蛋白 | |
CN107739410B (zh) | CD3单链抗体-iRGD融合蛋白、制备及其作为抗肿瘤药物的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180720 |
|
RJ01 | Rejection of invention patent application after publication |