CN108732153B - Method for detecting biological activity of bovine interferon α - Google Patents
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Abstract
The invention discloses a method for detecting biological activity of bovine interferon α and application thereof, wherein the method comprises the following steps of obtaining a Mxp gene fragment of bovine Mx protein by adopting PCR amplification, removing pCMV of pEGFP-N1 carrier plasmid, replacing pCMV in original pEGFP-N1 carrier plasmid by the Mxp gene fragment of the bovine Mx protein obtained by the PCR amplification through T4DNA ligase, constructing pEGFP-N1-Mxp plasmid, transfecting cells by the pEGFP-N1-Mxp plasmid, screening out stable transfected cell strains through neomycin, carrying out cloning culture on the screened stable transfected cell strains, adding bovine interferon α to incubate with the stable transfected cell strains after the cloning culture, activating the activity of an Mx gene promoter to promote the expression of EGFP in cells, and positively correlating the fluorescence intensity of the cells after irradiation of an excitation light source with the biological activity of the bovine interferon α, so that the biological activity of the bovine interferon α can be quantitatively evaluated.
Description
Technical Field
The invention relates to a method for detecting the biological activity of bovine interferon α, belonging to the technical field of interferon activity detection.
Background
Interferons (IFNs) are broad-spectrum antiviral glycoproteins secreted by recipient cells following viral infection of cells and organisms, or action of nucleic acids, bacterial endotoxins, mitogens and the like, and are classified into type I, type II and type III interferons, depending on their source and physicochemical properties, type I interferons include IFN- α - β,
and IFN-tau, type II interferons, i.e., IFN-gamma, type III interferons, i.e., I L-28A, I L-28B, and I L-29.
IFN- α has four main functions, namely, firstly, the cells infected with virus and the surrounding cells can enter an endogenous antiviral state to limit the spread of the virus, secondly, the natural immune response is maintained in a balanced state to promote the antigen presentation and the NK cell function and simultaneously inhibit the proinflammatory signal path and the cytokine production, thirdly, the adaptive immune system is activated to promote the generation of high-affinity antigen-specific T cells and promote the B cell response and the immune memory, and fourthly, the effect of inhibiting the virus proliferation is achieved.
The mechanism of action of IFN- α was elucidated by 25 years ago, IFN binding to receptors activates receptor-associated JAK1 and TYK2, phosphorylates STAT1 and STAT2 tyrosine, phosphorylated STAT1 and STAT2 form dimers and translocates into the nucleus, IFN-stimulating factor 3(ISGF3) assembling IFN-regulatory factor 9(IRF9) to form a 3-mer, ISGF3 binds to its cognate DNA sequence (IFN-stimulated response elements, ISREs) to directly activate ISGs transcription, bringing host cells into an antiviral state.
IFN- α can activate Mx promoter (Mx promoter, Mxp) through JAK-STAT signal transduction pathway, promote the expression of Mx, Mx can inhibit the replication of minus-strand RNA virus, two proteins Mx and Mx2 exist in cattle, wherein Mx plays a main role, Mxp has better specificity, can not be started by other cytokines such as interleukin and tumor necrosis factor, and can accurately reflect the biological function of IFN- α.
The method mainly comprises 3 methods for detecting the biological activity of the interferon, namely virus replication inhibition, plaque reduction analysis and cytopathic inhibition, but the 3 methods are easy to generate larger errors, poor in repeatability and low in accuracy, recently, a Mxp-L uciferase (luciferase) system is used for detecting the biological activity of the type I interferon, the system detects the capability of IFN activating Mxp without false positive, the whole process completely avoids the use of VSV and other live viruses without the concern of biological safety, the detection procedure is simplified, the time is greatly shortened, the detection can be completed within about 6 hours after IFN processing cells, the detection speed is high, the expression of the luciferase can be accurately quantified through an instrument, the accuracy of IFN quantification is improved, the high-flux operation of a large number of samples is facilitated, but the method is based on plasmid instant transfection, an internal reference plasmid needs to be prepared each time, the plasmid transfection efficiency is guaranteed to be consistent, and an accurate detection result can be obtained, so that the development in a common laboratory is difficult, a substrate is needed, and the use cost of the luciferase is increased.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for detecting the biological activity of bovine interferon α by using green fluorescent protein (EGFP), which does not need an internal reference plasmid, can simplify the detection process, does not need repeated plasmid transfection, improves the accuracy and the repeatability and has stronger practicability.
The purpose of the invention is realized by the following technical scheme:
a method for detecting the biological activity of bovine interferon α comprises the following steps:
adopting PCR amplification to obtain Mxp gene segment of the Mx protein of the cattle;
replacing a gene fragment of pCMV in a plasmid of an original pEGFP-N1 vector by using a gene fragment of Mxp of the bovine Mx protein obtained by PCR amplification through T4DNA ligase to construct a pEGFP-N1-Mxp plasmid;
transfecting cells by using pEGFP-N1-Mxp plasmid, and screening out a stable transfected cell strain by using neomycin;
cloning and culturing the screened stable transfected cell strain;
preparing a standard curve by using a bovine interferon α standard substance diluted in a gradient manner, and determining a mathematical logic relation between interferon titer and a fluorescence value;
adding a bovine interferon α sample to be detected into cells after cloning culture, detecting fluorescence intensity, and evaluating the titer of the bovine interferon α sample to be detected by combining a standard curve.
In the method for detecting the biological activity of the bovine interferon α, a bovine interferon α sample to be detected is added into cells after cloning culture, and incubation needs to be carried out for a certain time, generally about 6 hours.
In the method for detecting the biological activity of the bovine interferon α, the bovine interferon α is added into a stable transfected cell strain, the activity of an Mx gene promoter is activated to promote the expression of EGFP in cells, and the intensity of fluorescence emitted by the cells after irradiation of an excitation light source is positively correlated with the biological activity of the bovine interferon α, so that the biological activity of the bovine interferon α can be quantitatively evaluated.
In the present invention, Mxp refers to bovine Mx gene promoter region, and pCMV refers to CMV promoter region of pEGFP-N1 plasmid.
In the method for detecting the biological activity of the bovine interferon α, preferably, the step of obtaining the Mxp gene fragment of the bovine Mx protein by PCR amplification comprises:
according to the gene sequence of bovine Mx protein published in Genebank, a promoter region containing ISRE reaction elements at the 5' end is selected, PCR primers are designed, and PCR amplification is carried out;
then, after double enzyme digestion, the Mxp gene fragment is obtained by gel cutting, recovery and purification.
In the above-mentioned method for detecting the biological activity of bovine interferon α, the method for designing PCR primers is conventional.
In the method for detecting the biological activity of the bovine interferon α, preferably, the step of constructing the plasmid pEGFP-N1-Mxp by replacing the gene fragment of pCMV in the plasmid of pEGFP-N1 by T4DNA ligase with the gene fragment of Mxp of the bovine Mx protein obtained by PCR amplification comprises the following steps:
removing the gene fragment of pCMV of the pEGFP-N1 carrier plasmid;
replacing the gene fragment of pCMV in the original pEGFP-N1 vector plasmid by the gene fragment of Mxp through T4DNA ligase to construct a recombinant plasmid pEGFP-N1-Mxp;
transforming Escherichia coli DH5 α competent cells, shaking, extracting recombinant plasmids, obtaining DNA sequences of Mxp fragments of the recombinant plasmids through DNA sequencing, and selecting the recombinant plasmids with correct sequences;
the sequence is shown as sequence 1.
In the method for detecting the biological activity of the bovine interferon α, preferably, the steps of transfecting cells with pEGFP-N1-Mxp plasmid and selecting a stable transfected cell strain by neomycin comprise:
the pEGFP-N1-Mxp plasmid is transfected to cells through a transfection reagent, after about 96 hours of transfection, the cells are changed into a complete culture medium containing neomycin of about 2 mu g/m L for continuous culture for 14 days, and the cells with neomycin resistance are screened out, namely the cells with stable transfection.
In the method for detecting the biological activity of the bovine interferon α, preferably, the cloning culture is to culture the selected stably transfected cell line by a limiting dilution culture method, so as to obtain a clone formed by a single cell.
In the method for detecting the biological activity of bovine interferon α, the step of cloning and culturing the selected stably transfected cell line preferably further comprises detecting the cloned cell, and identifying whether the cell genome contains Mxp gene by PCR.
In the method for detecting the biological activity of the bovine interferon α, preferably, the sequence of the Mxp gene fragment is shown as sequence 1.
In the method for detecting the biological activity of the bovine interferon α, the method for removing the pCMV of the pEGFP-N1 vector plasmid is preferably a double-enzyme cleavage method.
In the method for detecting the biological activity of the bovine interferon α, the cells are preferably MDBK cells (bovine kidney cells).
In the method for detecting the biological activity of bovine interferon α, preferably, the bovine interferon α standard substance is a recombinant bovine α interferon prepared by a preparation method of a recombinant bovine α interferon standard substance disclosed in the patent with publication number CN 106319006A.
The invention also provides application of the bovine interferon α biological activity detection method in biological activity detection of natural or recombinant bovine interferon α.
The invention constructs a reporter gene plasmid which takes a cattle Mx gene promoter as a promoter to start EGFP expression, transfects MDBK cells, screens stable transfected cell strains, and uses a recombinant bovine interferon α standard product and a sample to be detected which are diluted in a gradient manner to be respectively incubated with the cells, detects the luminous value of the EGFP in the cells, and uses the recombinant bovine interferon α standard product to prepare a standard curve to detect the biological activity of the bovine interferon α in the sample to be detected.
The invention has the outstanding effects that:
according to the invention, the Mxp-EGFP reporter gene system is constructed by using green fluorescent protein (EGFP) to replace luciferase, and an internal reference plasmid is not required in the system, so that the Mxp-EGFP reporter gene system can be stably expressed in cells, and due to the improvement of a detection technology, the EGFP in the cells can be quantitatively detected, so that the interferon activity detection can be completed only by culturing the cells at each time and detecting the fluorescence intensity under a specific instrument after the cells are treated by interferon- α for 6 hours, therefore, the detection process is greatly simplified, the detection time is shortened, the accuracy is improved, and the system is more practical and suitable for being applied to large-scale production.
Drawings
FIG. 1 is a schematic diagram of the plasmid construction of pEGFP-N1-Mxp of example 1;
FIG. 2 is a standard graph of the EGFP reporter gene method of example 1;
FIG. 3 is a graph showing a comparison of the correlation between the EGFP reporter gene method of example 2 and the results of the trace cytopathy suppression test.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The materials used in the following examples of the invention are as follows:
MDBK cells (ATCCNO. CC L-22) were purchased from ATCC, VSV was provided by the institute for Wuhan virus, Chinese academy of sciences, eukaryotic expression vector pEGFP-N1 was purchased from Clontech, various restriction enzymes, Ex-Taq enzyme, T4DNA ligase and plasmid extraction kits were purchased from Takara, transfection reagent Fugen6 was purchased from Promega, Opti-MEM, DMEM medium and FBS were purchased from Invitrogen, various specifications of cell culture plates were purchased from Corning, multifunctional microplate reader was purchased from MD, and luciferase detection kit was purchased from Promega;
bovine interferon α standard (10)7IU/m L) is a recombinant bovine α interferon prepared by the method for preparing a recombinant bovine α interferon standard disclosed in the patent publication No. CN106319006A (the mode of example 1 or 2 thereof was selected).
Example 1
The present embodiment provides a method for detecting the biological activity of bovine interferon α, which is an application of detecting the biological activity of recombinant bovine interferon α, and the detection method of the present embodiment can also be correspondingly used for detecting the activity of natural bovine interferon α, and comprises the following steps:
selecting a promoter region containing ISRE reaction elements at the 5' end according to a gene sequence of bovine Mx protein published in Genebank, designing a PCR primer, extracting DNA from MDBK cells by a phenol-chloroform-isoamyl alcohol method to be used as a template, and performing PCR amplification by using the PCR primer and Ex-Taq enzyme (the underlined part is the position of an upstream primer and a downstream primer);
performing double enzyme digestion on a product obtained by PCR amplification through Ase I and Age I, and then performing gel cutting recovery and purification (an AseI enzyme digestion site is introduced into an upstream PCR primer, an Age I enzyme digestion site is introduced into a downstream PCR primer, wherein interferon reaction elements contained in the upstream PCR primer are shown in a bold letter part), so as to obtain a gene fragment of Mxp, wherein the sequence of the gene fragment of Mxp is shown as sequence 1;
removing the gene fragment of pCMV of the pEGFP-N1 carrier plasmid by adopting a double enzyme digestion method;
the Mxp gene fragment obtained by recovering the above-mentioned gel-cutting is substituted for pCMV gene fragment in pEGFP-N1 plasmid by T4DNA ligase, DH5 α competent cell is transformed, bacteria are shaken, positive clone is screened out, then plasmid is extracted, DNA sequence of Mxp is obtained by DNA sequencing, the plasmid with correct sequence is selected, namely pEGFP-N1-Mxp plasmid, and its construction schematic diagram is shown in figure 1.
Transfecting cells with pEGFP-N1-Mxp plasmid, and screening out a stable transfected cell strain sample through neomycin, namely extracting pEGFP-N1-Mxp plasmid by adopting an endotoxin-removing plasmid extraction kit, taking 1 mu g of plasmid, putting the 1 mu g of plasmid into a 1.5m L sterile EP tube, supplementing the volume to 500 mu l by using OptiMEM (OptiMEM medium), gently mixing the plasmids, incubating the plasmids at room temperature for 5min, taking 1.5m L sterile EP tube, adding 5 mu l of Fugen6 into the 500 mu l of serum-free Opti-MEM medium, gently mixing the plasmids, incubating the plasmid solution and the transfection reagent solution at room temperature for 20min, and using bloodless plasmid to perform incubation at room temperature for 5minSubculturing MDBK cells in clear Opti-MEM Medium into 6-well plates, 5 × 10 per well5(ii) individual cells; plasmid-transfection reagent complexes were added to each well at 37 ℃ with 5% CO2Culturing overnight in an incubator, removing the culture medium the next day, replacing with DMEM (containing sodium pyruvate and unnecessary amino acids) complete culture medium, continuing to culture for 96h, then using complete culture medium containing 2 mug/m L neomycin to screen and culture for 14 days, replacing fresh culture medium every 2 days, determining whether to passage according to the growth condition of the cells, wherein the cells surviving 14 days after neomycin action are stably transfected cells;
the cells obtained by screening for neomycin resistance were diluted to a cell concentration of 10/m L in complete medium containing 0.25. mu.g/m L puromycin, and 0.1m L cell suspension was added to each well of a 96-well plate containing 5% CO at 37 ℃2Culturing in an incubator; observing every day, selecting a hole with a single clone, culturing until the cell covers the surface area of the hole 1/3, transferring the cell into a 6-well plate, carrying out amplification culture by a limiting dilution culture method, identifying whether the cell genome contains Mxp genes or not by PCR, and freezing and preserving the seed, wherein the seed is named MDBK-pEGFP-N1-Mxp.
Taking bovine interferon α standard substance 1m L according to the ratio of 1:10, 1:100 and 1:103…1:107The gradient of (a) is diluted with complete medium;
inoculating 0.1m L stable transfected cell strain MDBK-pEGFP-N1-Mxp into 96-well cell culture plate, culturing overnight to make cell density reach 90%, discarding culture medium, adding 0.1m L recombinant bovine interferon α standard substance diluted with complete culture medium into each well, setting wells without recombinant bovine interferon α as control, and adding 5% CO at 37 deg.C2Continuously culturing for 6h in the incubator, taking out the culture plate, placing on a detection table of a multifunctional microplate reader, setting the wavelength of excitation light to be 488nm, the wavelength of acceptance to be 597nm, and the irradiation of a bottom light source, wherein the detection result is shown in figure 2, a standard curve is obtained, and the result shows that the fluorescence intensity gradually decreases along with the gradient dilution of the recombinant bovine interferon α, and the fluorescence intensity and the negative logarithm of the dilution of a recombinant bovine interferon α standard substance form a remarkable exponential relation R20.9943, the detection range is 0.1IU-107IU/mL。
Adding a bovine interferon α sample to be detected into cells after cloning culture, detecting fluorescence intensity, and evaluating by combining a standard curve to obtain the titer of the bovine interferon α sample to be detected.
Therefore, the detection method constructed by the invention does not need an internal reference plasmid, has good repeatability and is not influenced by other cell factors.
Example 2
This example provides a correlation experiment comparing the results of the method for detecting bovine interferon α biological activity with the results of the method for detecting a microcytopathic lesion.
And (3) detecting 20 parts of recombinant bovine interferon α samples simultaneously by adopting an EGFP (enhanced green fluorescent protein) reporter gene method and a micro cytopathy inhibition method, and analyzing whether the results of the two methods are related or not by linear regression.
The EGFP reporter gene detection protocol is shown in example 1.
The method for inhibiting microcytopathia comprises collecting bovine interferon α specimen 1m L, diluting with complete culture medium 1:100, diluting with complete culture medium at multiple ratio, subculturing MDBK, inoculating to 96-well cell culture plate, inoculating 100 μ l cell suspension (2 × 10) per well5M L), adding α 100. mu.l of recombinant bovine interferon (at different dilutions) per well, adding 100. mu.l of culture medium to virus control and cell control, and adding 5% CO at 37 ℃2Continuously culturing for 16h in the incubator; the medium was discarded and 100. mu.l of VSV (VSV virus titer capable of complete shedding of MDBK cells at 24 h) was added to each well, except for the cell control wells, at 37 ℃ with 5% CO2And (3) continuously culturing for 24h in the incubator, observing the cell breakage of the virus control hole to 100% under a microscope, and when the cell control is normal, staining the cell by using crystal violet, observing and recording by naked eyes after washing, wherein the cells in the cell control hole are completely purple, the virus control is not stained (100% CPE + + +) to indicate that the system is established, observing the staining conditions in different holes on the basis of the staining conditions, recording the CPE conditions, and calculating the dilution multiple of half cytopathic inhibition by using a Reed-Muench method, thereby obtaining the titer of the interferon.
The results are shown in FIG. 3, and correlation analysis of the two detection methods shows R2Greater than 0.9, indicating twoThe result shows that the interferon activity detection method established by the invention is simple and quick, has reliable and stable detection result, and can replace a trace cytopathy inhibition method, namely the traditional detection method, to be applied to the activity determination of natural or recombinant bovine interferon α.
In conclusion, the embodiment of the invention utilizes green fluorescent protein (EGFP) to replace luciferase to construct Mxp-EGFP reporter gene system, and the system does not need internal reference plasmid, so that Mxp-EGFP reporter gene system can be stably expressed in cells, and due to the improvement of detection technology, the EGFP in the cells can be quantitatively detected, and the detection of the antiviral activity of the interferon can be completed only by culturing the cells each time and detecting the fluorescence intensity under a specific instrument after the cells are treated by the interferon- α for 6 hours, so that the detection process is greatly simplified, the detection time is shortened, the accuracy is improved, and the system is more practical and suitable for being applied to large-scale production.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Sequence listing
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Claims (9)
1. A method for detecting the biological activity of bovine interferon α comprises the following steps:
adopting PCR amplification to obtain Mxp gene segment of the Mx protein of the cattle;
replacing a gene fragment of pCMV in a plasmid of an original pEGFP-N1 vector by using a gene fragment of Mxp of the bovine Mx protein obtained by PCR amplification through T4DNA ligase to construct a pEGFP-N1-Mxp plasmid;
transfecting cells by using pEGFP-N1-Mxp plasmid, and screening out a stable transfected cell strain by using neomycin;
cloning and culturing the screened stable transfected cell strain;
preparing a standard curve by using a bovine interferon α standard substance diluted in a gradient manner, and determining a mathematical logic relation between interferon titer and a fluorescence value;
adding a bovine interferon α sample to be detected into cells after cloning culture, detecting fluorescence intensity, and evaluating the titer of the bovine interferon α sample to be detected by combining a standard curve.
2. The method for detecting the biological activity of bovine interferon α according to claim 1, wherein the step of obtaining the Mxp gene fragment of bovine Mx protein by PCR amplification comprises:
according to the gene sequence of bovine Mx protein published in Genebank, a promoter region containing ISRE reaction elements at the 5' end is selected, PCR primers are designed, and PCR amplification is carried out;
then, after double enzyme digestion, the Mxp gene fragment is obtained by gel cutting, recovery and purification.
3. The method for detecting the biological activity of bovine interferon α according to claim 1, wherein the step of constructing the plasmid pEGFP-N1-Mxp by replacing the gene fragment of pCMV in the plasmid pEGFP-N1 vector with T4DNA ligase using the gene fragment of Mxp of the bovine Mx protein obtained by PCR amplification comprises:
removing the gene fragment of pCMV of the pEGFP-N1 carrier plasmid;
replacing the gene fragment of pCMV in the original pEGFP-N1 vector plasmid by the gene fragment of Mxp through T4DNA ligase to construct a recombinant plasmid pEGFP-N1-Mxp;
transforming Escherichia coli DH5 α competent cells, shaking, extracting recombinant plasmids, obtaining DNA sequences of Mxp fragments of the recombinant plasmids through DNA sequencing, and selecting the recombinant plasmids with correct sequences;
the correct sequence is shown in sequence 1.
4. The method for detecting the biological activity of bovine interferon α according to claim 1, wherein the step of transfecting cells with pEGFP-N1-Mxp plasmid and selecting stable transfected cell lines by neomycin comprises:
the pEGFP-N1-Mxp plasmid is transfected to cells through a transfection reagent, after transfection is carried out for 96 hours, the cells are changed into a complete culture medium containing 2 mu g/m L neomycin and continuously cultured for 14 days, and the cells with neomycin resistance are screened out, namely the cells are stably transfected.
5. The method for detecting the biological activity of bovine interferon α according to claim 1, wherein the cloning culture is performed by culturing the selected stably transfected cell line by limiting dilution culture, thereby obtaining a clone formed by a single cell;
preferably, the step of cloning and culturing the selected stably transfected cell line further comprises detecting the cloned cells and identifying whether the Mxp gene is contained in the cell genome by PCR.
6. The method for detecting the biological activity of bovine interferon α according to claim 1 or 2, wherein the sequence of Mxp gene fragment is shown as sequence 1.
7. The method of claim 3, wherein the pCMV removal method using pEGFP-N1 vector plasmid is a double-enzymatic cleavage method.
8. The method of claim 1 or 4, wherein the cells are MDBK cells.
9. Use of the bovine interferon α according to any of claims 1 to 8 for biological activity assay of natural or recombinant bovine interferon α.
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| CN103403166A (en) * | 2010-11-30 | 2013-11-20 | 株式会社Lg生命科学 | Novel hybrid promoter and recombinant vector comprising the same |
| CN104619726A (en) * | 2012-03-23 | 2015-05-13 | 苏州鲲鹏生物技术有限公司 | Fusion proteins of superfolder green fluorescent protein and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103403166A (en) * | 2010-11-30 | 2013-11-20 | 株式会社Lg生命科学 | Novel hybrid promoter and recombinant vector comprising the same |
| CN104619726A (en) * | 2012-03-23 | 2015-05-13 | 苏州鲲鹏生物技术有限公司 | Fusion proteins of superfolder green fluorescent protein and use thereof |
Non-Patent Citations (4)
| Title |
|---|
| Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model;Farahnaz Motamedi Sedeh et al;《VIROLOGICA SINICA》;20121031;286-291 * |
| Recombinant Interferon-c Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo;Ling Zhang et al;《PLOS one》;20120831;第7卷(第8期);1-6 * |
| 基于Mx 启动子和荧光素酶报告基因定量检测鸡Ⅰ型干扰素生物学活性的研究;陈伟业等;《畜牧兽医学报》;20081231;第39卷(第1期);123-128 * |
| 重组杆状病毒表达牛β 干扰素及其生物学活性研究;陈伟业等;《微生物学报》;20071204;第47卷(第6期);992-996 * |
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| CN108732153A (en) | 2018-11-02 |
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