CN105396123A - Application of ovR2E in curative effect of HBV infection treatment - Google Patents

Application of ovR2E in curative effect of HBV infection treatment Download PDF

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Publication number
CN105396123A
CN105396123A CN201510810173.4A CN201510810173A CN105396123A CN 105396123 A CN105396123 A CN 105396123A CN 201510810173 A CN201510810173 A CN 201510810173A CN 105396123 A CN105396123 A CN 105396123A
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China
Prior art keywords
ovr2e
albumen
hifn
curative effect
hr2a
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CN201510810173.4A
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Chinese (zh)
Inventor
余治健
邓启文
陈重
程航
邓向斌
李多云
郑金鑫
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SHENZHEN CITY NANSHAN DISTRICT PEOPLE'S HOSPITAL
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SHENZHEN CITY NANSHAN DISTRICT PEOPLE'S HOSPITAL
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Priority to CN201510810173.4A priority Critical patent/CN105396123A/en
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Abstract

The invention provides application of ovR2E in a curative effect of HBV infection treatment. The application of the ovR2E in the curative effect of HBV infection treatment is characterized in that the ovR2E protein is combined with hIFN-alpha to promote combination of a ligand and hIFNA-2c, and the ligand inhibition HBV replication activity is strengthened. According to the technical scheme, the ovR2E protein improves the hIFN-alpha inhibition HBV replication level, hR2a protein does not have strengthening and regulating effects on ligand inhibition HBV replication action, the ovR2E protein can be combined with the hIFN-alpha and strengthen the ligand inhibition HBV replication activity, a foundation is laid for the situation that the ovR2E protein and the hIFN-alpha are prepared into a mixture used for improving the curative effect of the hIFN-alpha on treating CHB in future, and a new approach is provided for treating the CHB.

Description

The application of ovR2E albumen curative effect in HBV infection treatment
Technical field
The present invention relates to technical field of molecular biology, particularly the application of ovR2E albumen curative effect in HBV infection treatment.
Background technology
Hepatitis B virus (hepatitisBvirus) is the DNA viruses causing mankind's acute hepatitis, chronic hepatitis, nowadays, and HBV infection serious threat human health, liver failure, liver cirrhosis and hepatocarcinoma that the whole world about has more than 100 ten thousand people to die from HBV infection to cause every year.
At present, IFN-α is current Treatment chronic Hepatitis B (chronichepatitisB, CHB) one of the first-line drug of patient, although long-acting IFN-α application improves curative effect of medication and compliance in recent years, no matter but be adjustment consumption, select different molecular hypotype or extend patient procedure, IFN-α treats CHB response rate and still only has 30-50%, long term, activity ratio was again still high, and therefore how improving IFN-α inhibition HBV replication activity and improving its antiviral therapy effect is one of current urgent clinical problem.
HumanIFN-α (humanIFN-α, hIFN-α) by with people I type interferon receptors (humantypeIInterferonreceptor, be called for short hIFNAR) membranous type subunit (comprising hIFNAR1 and hIFNAR-2c) combination formation complex transfer sell signal suppressing hepatitis B virus (HepatitisBvirus, HBV) copy, therefore affecting that part and Cell model subunit complex formed is one of potential strategy changing hIFN-α antiviral activity.
Summary of the invention
For above technical problem, the invention discloses ovR2E(extracellulardomainofovinetypeIinterferonreceptor, sheep I type interferon receptors subunit 2 extracellular region section) albumen application of curative effect in HBV infection treatment, inventor is found by great many of experiments, ovR2E albumen can be combined with humanIFN-α and strengthen part inhibition HBV replication activity, improves the curative effect of HBV infection treatment.
To this, technical scheme of the present invention is:
The application of ovR2E albumen curative effect in HBV infection treatment, after ovR2E albumen is combined with hIFN-α, promotes that part is combined with hIFNA-2c, strengthens part inhibition HBV replication active.
As a further improvement on the present invention, ovR2E albumen is directly combined with hIFN-α, strengthens hIFN-α to the activation of downstream MxA gene promoter.
As a further improvement on the present invention, it is active that ovR2E albumen significantly improves hIFN-α inhibition HBV replication, promotes that part is in cell and the downward effect of supernatant HBVDNA level.
As a further improvement on the present invention, hR2a albumen has negativity regulating action to the effect of part inhibition HBV replication.
As a further improvement on the present invention, ovR2E albumen is for the preparation of the mixture improving IFN-α treatment CHB curative effect.
Further, ovR2E albumen is combined the mixture for the preparation of improving IFN-α and treat CHB curative effect with hIFN-α.
IFN-α is one of first-line drug of current Treatment chronic Hepatitis B patient, although long-acting IFN-α application improves curative effect of medication and compliance in recent years, no matter but be adjustment consumption, select different molecular hypotype or extend patient procedure, IFN-α treats CHB response rate and still only has 30-50%, long term, activity ratio was again still high, and therefore how improving IFN-α inhibition HBV replication activity and improving its antiviral therapy effect is one of current urgent clinical problem.Our research work proves that ovR2E albumen can be combined with hIFN-α and improve Cell Interferon stimulated gene transcriptional activity first, raise part inhibition HBV replication level, HBV active mechanism is suppressed because OvR2E albumen can improve hIFN-α, therefore followingly mixture may be prepared into IFN-α, for improving the curative effect of interferon therapy CHB, there are nearly 9,600 ten thousand HBV infection patients in China, and therefore this application prospect has theoretical and realistic meaning preferably.
Beneficial effect of the present invention is: ovR2E albumen improves hIFN-α inhibition HBV replication level, hR2a albumen to the effect of part inhibition HBV replication without enhancing regulating action, ovR2E albumen can be combined with IFN-α and strengthen part inhibition HBV replication activity, lay a good foundation, for CHB treatment provides new way for following ovR2E albumen and IFN-α are prepared into mixture to treat CHB curative effect for improving IFN-α.
Accompanying drawing explanation
Fig. 1 is an embodiment of the present invention protein expression and Binding experiment result, wherein:
A is the result that pET-28a-ovR2E prokaryotic expression carrier expresses His-ovR2E albumen; Wherein 1,2 refer to that bacterial cytoplasm and supernatant adopt westernblot testing result respectively
B is the result adopting pET-28a-hR2a prokaryotic expression carrier to express His-hR2a albumen; Wherein 1,2 refer to that bacterial cytoplasm and supernatant adopt westernblot testing result respectively.
C is protein expression figure after employing westernblot detection confirmation fusion rotein and enzyme action.
D is 32p-ovR2E albumen and hIFN-α carry out crosslinked association reaction through SDS-PAGE electrophoretogram.
E is 32p-hR2a albumen and hIFN-α carry out crosslinked association reaction through SDS-PAGE electrophoretogram.
Fig. 2 is that ovR2E raising hIFN-alpha active researchs and analyses result figure;
Wherein, A is to MxA gene promoter activity (uciferase activity relative quantification) impact analysis figure after different expression way acquisition hR2a albumen mixes with hIFN-α;
B is to MxA gene promoter activity (uciferase activity relative quantification) impact analysis figure after the ovR2E albumen of different expression way acquisition mixes with hIFN-α;
C is to hbv replication level in cell (adopting log10 value to compare analysis after quantitatively adopting qPCR to detect) impact analysis figure after ovR2E and hIFN-α mixes;
D is to culture supernatant hbv replication level (adopting log10 value to compare analysis after quantitatively adopting qPCR to detect) impact analysis figure after ovR2E and hIFN-α mixes;
E is to MxA gene promoter activity impact analysis figure after the mixing of ovR2E and hIFN-α different proportion;
F is to MxA gene promoter activity impact analysis figure after the mixing of hR2a and hIFN-α different proportion;
*, p<0.01 is compared between statistical analysis group.
Fig. 3 is the Southernblot analysis chart that ovR2E albumen improves hIFN-α inhibition HBV replication.
Detailed description of the invention
Below in conjunction with accompanying drawing, preferably embodiment of the present invention is described in further detail.
Vivoexpression, purification obtain ovR2E albumen and complete itself and ligand binding assay:
(1) prokaryotic expression carrier and the protein purification research of sheep ovR2E and shIFNAR-2a is built:
(cell is numbered: SheepL1) total serum IgE to adopt trizol to extract sheep lung fibroblast-like cells, RT-PCR amplification sheep I type interferon receptors subunit 2 extracellular region section (extracellulardomainofovinetypeIinterferonreceptor, be called for short ovR2E) mRNA, its amplimer is:
ovR2ER：5-AGACGCGGATCCTCGTATGTTGTGCCTGATCT-3
ovR2EL：5-CCGCTAGAATTCTTATGTAGCAGGTTCTGATGACT-3，
The ovR2E genetic fragment of amplification is outer chief active region (27-246 the amino acids) (ProcNatlAcadSciUSA.2001 of sheep IFNAR2 born of the same parents; 98 (11): 6138-43), aminoacid sequence is as follows:
MLLSQNVSAIGPLNLYPMVHISLVFGISYVVPDLSDESCTLKMRFRNFQSILSWELKNRSIVPTHYTLWYTIMSKPEDMKVVKDCINITRSFCDLTDVWVNRTDMYISQVVGYRENAVVVSCMGSFFLASDKPLDPPKFEIVDFTNNISVNVKFRLDSPRIPSEELQFYLAFIEEHAGNSVKRHQPQITGNITENFNYVIDKLIPNTNYCISVYFEPK。
Build pGEXT-ovR2E and pET-28a-ovR2E prokaryotic expression carrier to be respectively used to express GST-ovR2E and His-ovR2E fusion rotein.For clear and definite human soluble I type interferon receptors sIFNAR2a(is called for short hR2a) activity get this albumen for contrast simultaneously, we construct pGEXT-hR2a and pET-28a-hR2a prokaryotic expression carrier for expressing GST-hR2a and His-hR2a fusion rotein.Adopt pGEXT-hR2a and pGEXT-ovR2E prokaryotic expression carrier vivoexpression gst fusion protein, adopt Thrombin treatment to obtain purification hR2a and ovR2E albumen.PET-28a-ovR2E and pET-28a-hR2a expressing protein westernblot is the results detailed in A and B in Fig. 1, wherein, pET-28a-ovR2E prokaryotic expression carrier expresses the A that His-ovR2E albumen the results are shown in Figure 1, employing pET-28a-hR2a prokaryotic expression carrier expression His-hR2a albumen the results are shown in Figure the B in 1, and wherein 1,2 refer to that bacterial cytoplasm and supernatant adopt westernblot testing result respectively.PGEXT-hR2a and pGEXT-ovR2E carries out expression gst fusion protein in vitro and obtains GST-hR2a and GST-ovR2E, hR2a and the ovR2E protein expression figure that another employing Thrombin treatment acquisition hR2a and ovR2E, GST-ovR2E and GST-hR2a albumen and Thrombin treatment purification obtain refers to the C in Fig. 1.For observing the combined function situation of hR2a albumen and ovR2E albumen and hIFN-α, adopting pGEX-2TK vector construction ovR2E albumen and hR2a protein expression vector, phosphorylation site being introduced to ovR2E albumen and hR2a albumen vivoexpression and is used for carrying out isotope 32p labelling. 32p-ovR2E albumen and 32p-hR2a albumen and hIFN-α carry out crosslinked association reaction and refer to D and E in Fig. 1 through SDS-PAGE electrophoretogram, both result promptings all can with hIFN-α, visible in the D in Fig. 1 32p-hR2a and hIFN-α is in conjunction with band and contrast 32p-hR2a; Visible in E in Fig. 1 32p-ovR2E and hIFN-α is in conjunction with band and contrast 32p-ovR2E.
(2) the external functional study that is combined with hIFN-α of ovR2E albumen:
Adopt pGEX-2TK vector construction ovR2E and hR2a protein expression vector, adopt isotope labeling reagent box (Pierce) to carry out labeled in vitro and purification respectively in the ovR2E albumen of purification and hR2a albumen, the same ProcNatlAcadSciUSA.2001 of method; 98 (11): 6138-43.Adopt 32p-ovR2E albumen and 32p-hR2a albumen carries out external crosslinked association reaction with hIFN-α respectively, and result is as shown in D and E in Fig. 1, visible, 32p-ovR2E albumen and 32p-hR2a albumen all can combine with hIFN-α.We observe simultaneously 32the association reaction of P-ovR2E albumen and hR2a albumen, both result displays directly do not combine, and prompting ovR2E albumen may not directly and hIFNAR-2c combination.
Adopt MxA gene promoter carrier pMxA-Luc transfection Huh7 cell, employing GST-ovR2E fusion rotein, ovR2E and His-ovR2E mix by 1:1 with hIFN-α respectively, and the various expressing protein of observation ovR2E mixes the impact on MxA gene promoter activity with hIFN-α; Separately get GST-hR2a, hR2a and His-hR2a albumen observe its mix with hIFN-α after impact on MxA gene promoter activity, affect interpretation of result figure as shown in A and B in Fig. 2, result shows, ovR2E and His-ovR2E all has facilitation to hIFN-alpha active, and hR2a has blocking effect to ligand activity.
Adopt Huh7 cell transfecting pUC19-1.24HBV, observe ovR2E to hIFN-α inhibition HBV replication activity influence, result is as shown in C and D in Fig. 2, it is active that C and D in comparison diagram 2 show that ovR2E can significantly improve hIFN-α inhibition HBV replication, and it can promote that part is in cell and the downward effect of supernatant HBVDNA level as seen.For clear and definite whether amount effect relationship, we carry out experiment in vitro observation after adopting different proportion mixing to ovR2E or hR2a and part, result, as shown in E and F in Fig. 2, all has facilitation to hIFN-alpha active from E and F in Fig. 2, ovR2E and His-ovR2E.
Comprehensive the above results, obtains ovR2E raising hIFN-α inhibition HBV replication hIFNAR by experiment in vitro and expresses closely-related PRELIMINARY RESULTS:
We are after Huh7 transit cell dye HBV expression vector, and after adopting ovR2E or hR2a albumen and ligand mixture to intervene, adopt Southernblot to detect HBVDNA level after each histone intervention, HBVDNA levels of replication the results are shown in Figure shown in 3.As seen from Figure 3, ovR2E albumen and ligand mixture intervention significantly reduce intracellular hbv replication level afterwards, and namely ovR2E albumen improves hIFN-α inhibition HBV replication level; And hR2a albumen has negativity regulating action to the effect of part inhibition HBV replication, illustrate that hR2a albumen mixes without this biological effect with part.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.

Claims (5)

  1. The application of 1.ovR2E albumen curative effect in HBV infection treatment, is characterized in that: after ovR2E albumen is combined with hIFN-α, promotes that part is combined with hIFNA-2c, strengthens part inhibition HBV replication active.
  2. 2. the application of ovR2E albumen according to claim 1 curative effect in HBV infection treatment, is characterized in that: ovR2E albumen is directly combined with hIFN-α, strengthens hIFN-α to the activation of downstream MxA gene promoter.
  3. 3. ovR2E albumen according to claim 1 application of curative effect in HBV infection treatment, is characterized in that: it is active that ovR2E albumen improves hIFN-α inhibition HBV replication, promotes that part is in cell and the downward effect of supernatant HBVDNA level.
  4. 4. the application of ovR2E albumen according to claim 1 curative effect in HBV infection treatment, is characterized in that: hR2a albumen has negativity regulating action to the effect of part inhibition HBV replication.
  5. 5. the application of the curative effect in HBV infection treatment of the ovR2E albumen according to claim 1 ~ 4 any one, is characterized in that: ovR2E albumen treats the mixture of CHB curative effect for the preparation of improving IFN-α.
CN201510810173.4A 2015-11-20 2015-11-20 Application of ovR2E in curative effect of HBV infection treatment Pending CN105396123A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1622957A (en) * 2001-12-31 2005-06-01 耶达研究发展有限公司 IFNAR2 mutants, their production and use
CN103536917A (en) * 2013-10-30 2014-01-29 苏州丁孚靶点生物技术有限公司 Use of interferon in treatment of tumor, and related product and method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1622957A (en) * 2001-12-31 2005-06-01 耶达研究发展有限公司 IFNAR2 mutants, their production and use
CN103536917A (en) * 2013-10-30 2014-01-29 苏州丁孚靶点生物技术有限公司 Use of interferon in treatment of tumor, and related product and method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAN CHUN-SHENG，ET AL.: "Antiviral activities of the soluble extracellular domains of type I interferon receptors", 《PNAS》 *
刘凤军 等: "干扰素直接抗乙型肝炎病毒作用机制研究的现状，", 《生物医学工程学杂志》 *

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