重组牛IFN-λ3的制备及其与IFN-α/IFN-β/IFN-γ抗病毒活性比较研究
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摘要
干扰素(Interferon，IFN)是由动物细胞产生的，具有抗病毒、抗肿瘤及免疫调节等作用的一类细胞因子。IFN分为I型、II型和新发现的III型，III型干扰素又称为IFN-λs，其氨基酸水平和蛋白质功能与I型相近。由于IFN-λs受体组织分布特异性，IFN-λs主要在呼吸道、消化道和皮肤黏膜组织以及上皮细胞或某些肿瘤细胞发挥抗病毒作用，与IFN-α相比，能够有效地降低毒副作用。在我国，奶牛病毒性传染病如口蹄疫（FMD）、牛病毒性腹泻病（BVD）和牛传染性鼻气管炎（IBR）等可通过消化道、呼吸道传播的疫病，至今危害仍比较严峻，因此牛IFN-λs成为继IFN-α和IFN-β后，抗病毒制剂研发的一个方向。为深入研究牛IFN-λs的抗病毒活性及其机制，揭示其用于防治牛病毒病的可行性，本研究利用毕赤酵母分泌表达系统表达了无冗余氨基酸的重组牛IFN-λ3，同时酵母分泌表达了重组牛IFN-α和IFN-β，大肠杆菌表达系统表达了重组牛IFN-β和IFN-γ，深入研究了各重组干扰素的抗病毒活性，比较了IFN-λ3与I型和II型干扰素单独或联合应用抗病毒活性的异同，主要研究内容如下：
1.首先在毕赤酵母表达系统实现了无冗余氨基酸重组牛IFN-λ3的可溶性表达。参考酵母密码子使用偏好性，同时考虑自由能和二级结构等要素，将已知的牛IFN-λ3（boIFN-λ3）基因序列优化成适合酵母表达的最优序列，然后利用重叠延伸PCR技术将设计的相互重叠20bp左右的14条寡核苷酸融合，获得boIFN-λ3基因和及其等位基因命名为boIFN-λ3*（1个基因差异使得成熟肽第18位氨基酸不同）。通过酶切连接的方式，成功构建酵母分泌表达载体pPICZαA-boIFN-λ3和pPICZαA-boIFN-λ3*。在毕赤酵母表达系统中通过一系列的筛选鉴定，如阳性重组酵母菌的鉴定，高表达酵母菌株的筛选，诱导表达条件的优化等最终表达了重组boIFN-λ3和boIFN-λ3*蛋白，表达量均可达1.2g/L，均以糖基化蛋白（23kDa大小，占总蛋白70%左右）和非糖基化蛋白（18kDa大小，占总蛋白15%左右）2种形式表达。经硫酸铵盐析和阳离子交换层析获得了纯化蛋白，重组蛋白均具有良好的抗原性，同时制备了抗boIFN-λ3*多抗。在MDBK-VSV系统测定重组boIFN-λ3和boIFN-λ3*蛋白的生物学效价分别为2.39±0.15×10~6U/mg/ml和2.15±0.40×10~6U/mg/ml。理化特性方面，重组蛋白均对胰酶敏感，对热较为不敏感（63℃），对酸碱部分敏感（pH2活性下降2倍），可被特异性抗体中和。从重组蛋白的表达、纯化、糖基化分析、理化特性、生物学活性和致细胞毒性等综合分析，重组蛋白boIFN-λ3和boIFN-λ3*没有区别。
2.表达纯化了重组牛IFN-α/IFN-β/IFN-γ并初步测定了它们的生物学活性。为比较重组boIFN-λ3与I型和II型IFN的抗病毒活性，酵母分泌表达系统表达了重组牛IFN-α和IFN-β，大肠杆菌系统表达纯化了重组牛IFN-β和IFN-γ，测定它们的生物学活性分别为1.50±0.98×10~7、1.25±0.38×10~4、2.44±0.91×10~3和0.81±0.21×10~5U/mg/ml。在MDBK-VSV系统中4种重组干扰素抗病毒效价大小排序为boIFN-α>boIFN-λ3> boIFN-γ>boIFN-β。在EBK和MDBK细胞上利用MTT法测定4种重组干扰素的细胞毒性，结果显示，重组boIFN-λ3和其他IFNs单独或联合应用致细胞毒性均较小。
3.重组boIFN-λ3和boIFN-α/boIFN-β/boIFN-γ诱导Mx1蛋白和ISRE启动子活性的研究。经过Western blot检测重组boIFN-λ3在4种上皮细胞源细胞（EBK、BT、MDBK和BMEC）中均能够诱导产生Mx1蛋白。采用双荧光素酶报告基因系统，通过检测ISRE报告基因的表达水平来间接反映干扰素的生物活性。结果显示不同浓度的重组IFNs诱导启动子表达均呈一定程度的剂量依赖性，重组boIFN-λ3和boIFN-γ诱导报告基因的表达水平呈时间依赖性，48h达到较高水平；而boIFN-α和boIFN-β能够快速的诱导报告基因表达，干扰素作用12h即达到较高的水平，且维持到48h。联合应用时部分组合比单独应用时诱导ISRE活性高。因此各IFNs刺激产生的ISRE启动子活性的差异可能是重组boIFN-λ3与其他IFNs协同抗病毒活性的机理之一。
4.比较研究了重组boIFN-λ3和重组boIFN-α/boIFN-β/boIFN-γ抗IBRV、FMDV和BVDV的研究。通过TCID50法测定抗IBRV和FMDV活性，抗cpBVDV采用噬斑减数法，抗ncpBVDV采用半定量RT-PCR法。结果显示重组boIFN-λ3和其他3种重组IFNs均具有抗IBRV和FMDV的活性，且呈一定程度的剂量和时间依赖性。其中抗FMDV活性较强，抗IBRV活性较弱。重组boIFN-λ3可条件性的抑制cpBVDV和ncpBVDV的增殖，即在先孵育干扰素后感染病毒时，重组boIFN-λ3可抑制BVDV增殖，反之则没有作用。
5.重组boIFN-λ3和boIFN-α/boIFN-β/boIFN-γ协同抗病毒的研究。通过测定干扰素作用后的病毒-细胞混悬液的TCID50，结果显示重组boIFN-λ3抗IBRV时和boIFN-γ协同作用强，抗FMDV时，boIFN-λ3在EBK和BT细胞上均显示出和boIFN-α/boIFN-β明显的协同作用，提示在临床应用时可以联合应用重组boIFN-λ3和其他干扰素。推测协同机制可能与ISRE启动子活化水平及干扰素刺激基因（ISGs）的表达等有关。
6. ncpBVDV持续性细胞感染是否影响重组boIFN-λ3抗病毒活性的研究。ncpBVDV（HLJ-11）感染MDBK细胞后在不同浓度重组boIFN-λ3和重组boIFN-α压力筛选下传代8次，通过半定量RT-PCR检测BVDV核酸，结果显示在干扰素压力下，病毒可以耐受干扰素，造成细胞持续性感染，而此种状态下的MDBK细胞仍能感染VSV，且不影响重组boIFN-λ3在此细胞上发挥抗VSV活性，因此这种细胞是耐受BVDV同时对IFN敏感的细胞。另一方面，ncpBVDV预先感染MDBK细胞不影响boIFN-λ3和其他IFNs在此细胞上发挥抗VSV和IBRV的作用。
因此，牛IFN-λ3蛋白的研制及其与I型和II型干扰素抗病毒活性的深入比较研究为进一步了解牛IFN-λs的生物学功能提供了理论基础并为抗病毒制剂的制备提供了物质材料。
IFNs are key cytokines in the establishment of the activities with antiviral, antitumor andimmunomodulatory activities. Three distinct types of IFNs are now recognized (type I, II, and III)based on their structural features, receptor usage and biological activities. The type III interferon isalso known as the IFN-λs and for the amino acid level and protein functions, IFN-λs are morerelated to type I IFNs. However, the more limited tissue expression of IFN-λ receptors suggeststhat type III IFNs do not simply recapitulate the type I IFN antiviral system. It has the antiviraleffects in the respiratory tract, gastrointestinal tract, skin mucosa, epithelial cells and some tumorcells, which can effectively reduce the toxic side effects. In our country, the foot-and-mouthdisease (FMD), bovine viral diarrhea disease (BVD) and infectious bovine rhinotracheitis (IBR)which spread through the digestive tract, respiratory tract is still relatively severe. Therecombinant bovine IFN-λs may play an important role in the control the diseases by its antiviralactivity. For in-depth reseach of antiviral activity of the bovine IFN-λs, revealing its feasibility forthe control of bovine viral disease, the non-redundant amino acid recombinant bovine IFN-λ3wassoluble expressed in Pichia pastoris. The recombinant bovine IFN-α and the recombinant bovineIFN-β was also expressed in Pichia pastoris. On the other hand, the recombinant bovine IFN-β andthe recombinant bovine IFN-γ was espressed from the E.coli expression system. In the direction offurther understand the biological function of bovine IFN-λ, the in-depth reseach of the antiviralactivities of the IFN-λ3was carried out and compared with type I and type II interferons alone orcombination used. The main contents are as follows:
1. The recombinant bovine IFN-λ3was soluble expression in Pichia pastoris. Taking into theyeast codon uasage, the free energy and the secondary structure of the bovine IFN-λ3, theoptimized bovine IFN-λ3sequence which named as boIFN-λ3and its allele (named as theboIFN-λ3*that one gene was different which caused the eighteen amina acid of the mature peptideof the bovine IFN-λ3was variability) was designed and the sequences were amplified using thegene splicing by overlap extension (SOE) with the synthesized14oligonucleotide. Then thesecretion expression vector pPICZαA-boIFN-λ3and pPICZaA-BoIFN-λ3*was successfullyconstructed. With the screening and identification of the positive recombinantGS115-pPICZαA-boIFN-λ3, the selection of the high expression stain and the optimization of the induced expression system, we expressed the recombinant boIFN-λ3and boIFN-λ3*with theexpression levels up to1.2g/L. The glycosylated protein (23kDa, about70%of the total protein)and non-glycosylated protein (18kDa, about15%of the total protein) was detected byglycoprotein staining. The protein was purified by the ammonium sulfate precipitation and thecation exchange chromatography. The recombinant protein has good antigenicity with thedeveloped polyclonal antibody. The antiviral activity of the recombinant boIFN-λ3and boIFN-λ3*was2.39±0.15×106U/mg/ml and2.15±0.40×106U/mg/ml in MDBK-VSV detecting system. Therecombinant proteins were inactivatied with the trypsin, not sensitive to heat(63℃) and partlysensitive to pH (pH2activity decreased by2times). With the comprehensive analysis, theexpression, purification, physical and chemical properties, biological activity and inducedcytotoxicity was no difference between the recombinant boIFN-λ3and the boIFN-λ3*.
2. The recombinant bovine IFN-α/IFN-β/IFN-γ was expressed, purified and preliminarydetermined their biological activity. For comparison antiviral activity of boIFN-λ3with type I andtype II IFN, The recombinant boIFN-α and the recombinant boIFN-β was expressed in Pichiapastoris and the biological activity was1.50±0.98×107U/mg/ml and1.25±0.38×104U/mg/ml. Thebiological activity of the prokaryotic expressed and purified recombinant boIFN-β and the boIFN-γwas2.44±0.91×103U/mg/ml and0.81±0.21×105U/mg/ml. The antiviral potency was therecombinant boIFN-α>boIFN-λ3>boIFN-γ>boIFN-β in MDBK-VSV system. Cytotoxicitymeasurements by MTT assay in EBK and MDBK cells showed that boIFN-λ3and other IFNsinduced lower cytotoxicity alone or in combination.
3. The Mx1protein and the ISRE promoter activity was induced by IFNs. The Mx1proteinwas induced by recombinant boIFN-λ3and other IFNs in epithelial cell-derived cells (EBK, BT,MDBK and BMEC). We used the ISRE (interferon-stimulated response element) luciferasereporter assay to assess activity downstream of the JAK-STAT signaling pathway. The resultsshowed that the every recombinant boIFN induced ISRE luciferase activity in a dose-dependentmanner; and the recombinant boIFN-λ3and boIFN-γ induced reporter gene expression level wastime-dependent and reached a high level at48h, while the recombinant boIFN-α and boIFN-βinduced ISRE luciferase more rapidly that reached the high level at12h and maintained to48h.The combination of the recombinant boIFN-λ3with other IFNs was high than alone except theboIFN-λ3plus the boIFN-γ.
4. The anti-FMDV/IBRV/BVDV was comparative research on the recombinant boIFN-λ3andboIFN-α/boIFN-β/boIFN-γ. The methods were about TCID50assay and the plaque reduction assay.The results showed that the recombinant boIFN-λ3and other three recombinant IFNs haveanti-IBRV and anti-FMDV activity, with a certain degree of dose and time-dependent manner. Theactivity of anti-IBRV was weak while anti-FMDV strong. The inhibitory effect of the recombinantboIFN-λ3on cytopathic BVDV (cpBVDV) and noncytopathic BVDV (ncpBVDV) replication wasobvious while treatment of MDBK cells with IFN before and in a dose-dependent manner. While treatment of MDBK cells with IFN after and the BVDV infecton first, any of the IFNsconcentration has no activity to inhibate the BVDV replication.
5. The synergistic antiviral research of the recombinant boIFN-λ3with the recombinantboIFN-α/boIFN-β/boIFN-γ. Using the cytopathic inhibition assay (TCID50assay), the virus-cellsuspension harvested from the MDBK or EBK cells treated before with the recombinant boIFN-λ3alone or combination with the boIFN-α/IFN-β/IFN-γ was assessed. In combination, therecombinant boIFN-λ3and the boIFN-γ had a synergistic anti-IBRV effect in MDBK cells, and therecombinant boIFN-λ3with the recombinant boIFN-α or boIFN-β could significantly inhibit theFMDV replication in EBK cells and BT cells. Thus, the synergistic anti-FMDV effect was apparentby combination with the recombinant boIFN-λ3and the boIFN-α/boIFN-β. It was prompted to bewidely used with the combination of the recombinant boIFN-λ3and other IFNs in clinicalapplications in the future. The synergistic enhancement was observed may relate to the ISREactivity or up regulation of the interferon stimulated genes (ISGs).
6. The impact on the activity of the recombinant boIFN-λ3when ncpBVDV persisitly infected.To determine whether ncpBVDV can be eliminated from the infected cell monolayer by prolongedinterferons treatment, we passaged ncpBVDV infected in MDBK cells8times in the presence ofvarious amounts of the recombinant boIFN-λ3, which the recombinant boIFN-α as the control.Using the semi-quantitative RT-PCR to detect BVDV nucleic acid, the cell culture passages ofMDBK cells infected with the HLI-11strain in the presence of up to100U/ml recombinantboIFN-λ3were not able to eliminate the virus infection，which was to say the BVDV can toleratethe interferon and the MDBK cells tolerate the BVDV, resulting in the persistent infection of cells.But this type cells was sensitive to VSV virus and the recombinant boIFN-λ3could prevent theinfection of the cell monolayer with VSV. This ncpBVDV pre-infected MDBK cells does not affectboIFN-λ3and other IFNs on to play anti-VSV and anti-IBRV effect.
In conclusion, the generation of the bovine IFN-λ3, the research of the antiviral activites ofthe recombinant boIFN-λ3and its synergistic effects with type I or type II interferons will help usto further understand the biological function of bovine IFN-λs. In addition, there is one moreantiviral agents was provided for future use.
1.首先在毕赤酵母表达系统实现了无冗余氨基酸重组牛IFN-λ3的可溶性表达。参考酵母密码子使用偏好性，同时考虑自由能和二级结构等要素，将已知的牛IFN-λ3（boIFN-λ3）基因序列优化成适合酵母表达的最优序列，然后利用重叠延伸PCR技术将设计的相互重叠20bp左右的14条寡核苷酸融合，获得boIFN-λ3基因和及其等位基因命名为boIFN-λ3*（1个基因差异使得成熟肽第18位氨基酸不同）。通过酶切连接的方式，成功构建酵母分泌表达载体pPICZαA-boIFN-λ3和pPICZαA-boIFN-λ3*。在毕赤酵母表达系统中通过一系列的筛选鉴定，如阳性重组酵母菌的鉴定，高表达酵母菌株的筛选，诱导表达条件的优化等最终表达了重组boIFN-λ3和boIFN-λ3*蛋白，表达量均可达1.2g/L，均以糖基化蛋白（23kDa大小，占总蛋白70%左右）和非糖基化蛋白（18kDa大小，占总蛋白15%左右）2种形式表达。经硫酸铵盐析和阳离子交换层析获得了纯化蛋白，重组蛋白均具有良好的抗原性，同时制备了抗boIFN-λ3*多抗。在MDBK-VSV系统测定重组boIFN-λ3和boIFN-λ3*蛋白的生物学效价分别为2.39±0.15×10~6U/mg/ml和2.15±0.40×10~6U/mg/ml。理化特性方面，重组蛋白均对胰酶敏感，对热较为不敏感（63℃），对酸碱部分敏感（pH2活性下降2倍），可被特异性抗体中和。从重组蛋白的表达、纯化、糖基化分析、理化特性、生物学活性和致细胞毒性等综合分析，重组蛋白boIFN-λ3和boIFN-λ3*没有区别。
2.表达纯化了重组牛IFN-α/IFN-β/IFN-γ并初步测定了它们的生物学活性。为比较重组boIFN-λ3与I型和II型IFN的抗病毒活性，酵母分泌表达系统表达了重组牛IFN-α和IFN-β，大肠杆菌系统表达纯化了重组牛IFN-β和IFN-γ，测定它们的生物学活性分别为1.50±0.98×10~7、1.25±0.38×10~4、2.44±0.91×10~3和0.81±0.21×10~5U/mg/ml。在MDBK-VSV系统中4种重组干扰素抗病毒效价大小排序为boIFN-α>boIFN-λ3> boIFN-γ>boIFN-β。在EBK和MDBK细胞上利用MTT法测定4种重组干扰素的细胞毒性，结果显示，重组boIFN-λ3和其他IFNs单独或联合应用致细胞毒性均较小。
3.重组boIFN-λ3和boIFN-α/boIFN-β/boIFN-γ诱导Mx1蛋白和ISRE启动子活性的研究。经过Western blot检测重组boIFN-λ3在4种上皮细胞源细胞（EBK、BT、MDBK和BMEC）中均能够诱导产生Mx1蛋白。采用双荧光素酶报告基因系统，通过检测ISRE报告基因的表达水平来间接反映干扰素的生物活性。结果显示不同浓度的重组IFNs诱导启动子表达均呈一定程度的剂量依赖性，重组boIFN-λ3和boIFN-γ诱导报告基因的表达水平呈时间依赖性，48h达到较高水平；而boIFN-α和boIFN-β能够快速的诱导报告基因表达，干扰素作用12h即达到较高的水平，且维持到48h。联合应用时部分组合比单独应用时诱导ISRE活性高。因此各IFNs刺激产生的ISRE启动子活性的差异可能是重组boIFN-λ3与其他IFNs协同抗病毒活性的机理之一。
4.比较研究了重组boIFN-λ3和重组boIFN-α/boIFN-β/boIFN-γ抗IBRV、FMDV和BVDV的研究。通过TCID50法测定抗IBRV和FMDV活性，抗cpBVDV采用噬斑减数法，抗ncpBVDV采用半定量RT-PCR法。结果显示重组boIFN-λ3和其他3种重组IFNs均具有抗IBRV和FMDV的活性，且呈一定程度的剂量和时间依赖性。其中抗FMDV活性较强，抗IBRV活性较弱。重组boIFN-λ3可条件性的抑制cpBVDV和ncpBVDV的增殖，即在先孵育干扰素后感染病毒时，重组boIFN-λ3可抑制BVDV增殖，反之则没有作用。
5.重组boIFN-λ3和boIFN-α/boIFN-β/boIFN-γ协同抗病毒的研究。通过测定干扰素作用后的病毒-细胞混悬液的TCID50，结果显示重组boIFN-λ3抗IBRV时和boIFN-γ协同作用强，抗FMDV时，boIFN-λ3在EBK和BT细胞上均显示出和boIFN-α/boIFN-β明显的协同作用，提示在临床应用时可以联合应用重组boIFN-λ3和其他干扰素。推测协同机制可能与ISRE启动子活化水平及干扰素刺激基因（ISGs）的表达等有关。
6. ncpBVDV持续性细胞感染是否影响重组boIFN-λ3抗病毒活性的研究。ncpBVDV（HLJ-11）感染MDBK细胞后在不同浓度重组boIFN-λ3和重组boIFN-α压力筛选下传代8次，通过半定量RT-PCR检测BVDV核酸，结果显示在干扰素压力下，病毒可以耐受干扰素，造成细胞持续性感染，而此种状态下的MDBK细胞仍能感染VSV，且不影响重组boIFN-λ3在此细胞上发挥抗VSV活性，因此这种细胞是耐受BVDV同时对IFN敏感的细胞。另一方面，ncpBVDV预先感染MDBK细胞不影响boIFN-λ3和其他IFNs在此细胞上发挥抗VSV和IBRV的作用。
因此，牛IFN-λ3蛋白的研制及其与I型和II型干扰素抗病毒活性的深入比较研究为进一步了解牛IFN-λs的生物学功能提供了理论基础并为抗病毒制剂的制备提供了物质材料。
IFNs are key cytokines in the establishment of the activities with antiviral, antitumor andimmunomodulatory activities. Three distinct types of IFNs are now recognized (type I, II, and III)based on their structural features, receptor usage and biological activities. The type III interferon isalso known as the IFN-λs and for the amino acid level and protein functions, IFN-λs are morerelated to type I IFNs. However, the more limited tissue expression of IFN-λ receptors suggeststhat type III IFNs do not simply recapitulate the type I IFN antiviral system. It has the antiviraleffects in the respiratory tract, gastrointestinal tract, skin mucosa, epithelial cells and some tumorcells, which can effectively reduce the toxic side effects. In our country, the foot-and-mouthdisease (FMD), bovine viral diarrhea disease (BVD) and infectious bovine rhinotracheitis (IBR)which spread through the digestive tract, respiratory tract is still relatively severe. Therecombinant bovine IFN-λs may play an important role in the control the diseases by its antiviralactivity. For in-depth reseach of antiviral activity of the bovine IFN-λs, revealing its feasibility forthe control of bovine viral disease, the non-redundant amino acid recombinant bovine IFN-λ3wassoluble expressed in Pichia pastoris. The recombinant bovine IFN-α and the recombinant bovineIFN-β was also expressed in Pichia pastoris. On the other hand, the recombinant bovine IFN-β andthe recombinant bovine IFN-γ was espressed from the E.coli expression system. In the direction offurther understand the biological function of bovine IFN-λ, the in-depth reseach of the antiviralactivities of the IFN-λ3was carried out and compared with type I and type II interferons alone orcombination used. The main contents are as follows:
1. The recombinant bovine IFN-λ3was soluble expression in Pichia pastoris. Taking into theyeast codon uasage, the free energy and the secondary structure of the bovine IFN-λ3, theoptimized bovine IFN-λ3sequence which named as boIFN-λ3and its allele (named as theboIFN-λ3*that one gene was different which caused the eighteen amina acid of the mature peptideof the bovine IFN-λ3was variability) was designed and the sequences were amplified using thegene splicing by overlap extension (SOE) with the synthesized14oligonucleotide. Then thesecretion expression vector pPICZαA-boIFN-λ3and pPICZaA-BoIFN-λ3*was successfullyconstructed. With the screening and identification of the positive recombinantGS115-pPICZαA-boIFN-λ3, the selection of the high expression stain and the optimization of the induced expression system, we expressed the recombinant boIFN-λ3and boIFN-λ3*with theexpression levels up to1.2g/L. The glycosylated protein (23kDa, about70%of the total protein)and non-glycosylated protein (18kDa, about15%of the total protein) was detected byglycoprotein staining. The protein was purified by the ammonium sulfate precipitation and thecation exchange chromatography. The recombinant protein has good antigenicity with thedeveloped polyclonal antibody. The antiviral activity of the recombinant boIFN-λ3and boIFN-λ3*was2.39±0.15×106U/mg/ml and2.15±0.40×106U/mg/ml in MDBK-VSV detecting system. Therecombinant proteins were inactivatied with the trypsin, not sensitive to heat(63℃) and partlysensitive to pH (pH2activity decreased by2times). With the comprehensive analysis, theexpression, purification, physical and chemical properties, biological activity and inducedcytotoxicity was no difference between the recombinant boIFN-λ3and the boIFN-λ3*.
2. The recombinant bovine IFN-α/IFN-β/IFN-γ was expressed, purified and preliminarydetermined their biological activity. For comparison antiviral activity of boIFN-λ3with type I andtype II IFN, The recombinant boIFN-α and the recombinant boIFN-β was expressed in Pichiapastoris and the biological activity was1.50±0.98×107U/mg/ml and1.25±0.38×104U/mg/ml. Thebiological activity of the prokaryotic expressed and purified recombinant boIFN-β and the boIFN-γwas2.44±0.91×103U/mg/ml and0.81±0.21×105U/mg/ml. The antiviral potency was therecombinant boIFN-α>boIFN-λ3>boIFN-γ>boIFN-β in MDBK-VSV system. Cytotoxicitymeasurements by MTT assay in EBK and MDBK cells showed that boIFN-λ3and other IFNsinduced lower cytotoxicity alone or in combination.
3. The Mx1protein and the ISRE promoter activity was induced by IFNs. The Mx1proteinwas induced by recombinant boIFN-λ3and other IFNs in epithelial cell-derived cells (EBK, BT,MDBK and BMEC). We used the ISRE (interferon-stimulated response element) luciferasereporter assay to assess activity downstream of the JAK-STAT signaling pathway. The resultsshowed that the every recombinant boIFN induced ISRE luciferase activity in a dose-dependentmanner; and the recombinant boIFN-λ3and boIFN-γ induced reporter gene expression level wastime-dependent and reached a high level at48h, while the recombinant boIFN-α and boIFN-βinduced ISRE luciferase more rapidly that reached the high level at12h and maintained to48h.The combination of the recombinant boIFN-λ3with other IFNs was high than alone except theboIFN-λ3plus the boIFN-γ.
4. The anti-FMDV/IBRV/BVDV was comparative research on the recombinant boIFN-λ3andboIFN-α/boIFN-β/boIFN-γ. The methods were about TCID50assay and the plaque reduction assay.The results showed that the recombinant boIFN-λ3and other three recombinant IFNs haveanti-IBRV and anti-FMDV activity, with a certain degree of dose and time-dependent manner. Theactivity of anti-IBRV was weak while anti-FMDV strong. The inhibitory effect of the recombinantboIFN-λ3on cytopathic BVDV (cpBVDV) and noncytopathic BVDV (ncpBVDV) replication wasobvious while treatment of MDBK cells with IFN before and in a dose-dependent manner. While treatment of MDBK cells with IFN after and the BVDV infecton first, any of the IFNsconcentration has no activity to inhibate the BVDV replication.
5. The synergistic antiviral research of the recombinant boIFN-λ3with the recombinantboIFN-α/boIFN-β/boIFN-γ. Using the cytopathic inhibition assay (TCID50assay), the virus-cellsuspension harvested from the MDBK or EBK cells treated before with the recombinant boIFN-λ3alone or combination with the boIFN-α/IFN-β/IFN-γ was assessed. In combination, therecombinant boIFN-λ3and the boIFN-γ had a synergistic anti-IBRV effect in MDBK cells, and therecombinant boIFN-λ3with the recombinant boIFN-α or boIFN-β could significantly inhibit theFMDV replication in EBK cells and BT cells. Thus, the synergistic anti-FMDV effect was apparentby combination with the recombinant boIFN-λ3and the boIFN-α/boIFN-β. It was prompted to bewidely used with the combination of the recombinant boIFN-λ3and other IFNs in clinicalapplications in the future. The synergistic enhancement was observed may relate to the ISREactivity or up regulation of the interferon stimulated genes (ISGs).
6. The impact on the activity of the recombinant boIFN-λ3when ncpBVDV persisitly infected.To determine whether ncpBVDV can be eliminated from the infected cell monolayer by prolongedinterferons treatment, we passaged ncpBVDV infected in MDBK cells8times in the presence ofvarious amounts of the recombinant boIFN-λ3, which the recombinant boIFN-α as the control.Using the semi-quantitative RT-PCR to detect BVDV nucleic acid, the cell culture passages ofMDBK cells infected with the HLI-11strain in the presence of up to100U/ml recombinantboIFN-λ3were not able to eliminate the virus infection，which was to say the BVDV can toleratethe interferon and the MDBK cells tolerate the BVDV, resulting in the persistent infection of cells.But this type cells was sensitive to VSV virus and the recombinant boIFN-λ3could prevent theinfection of the cell monolayer with VSV. This ncpBVDV pre-infected MDBK cells does not affectboIFN-λ3and other IFNs on to play anti-VSV and anti-IBRV effect.
In conclusion, the generation of the bovine IFN-λ3, the research of the antiviral activites ofthe recombinant boIFN-λ3and its synergistic effects with type I or type II interferons will help usto further understand the biological function of bovine IFN-λs. In addition, there is one moreantiviral agents was provided for future use.
引文
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