Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

Expression of Human Interferon α 2b Gene in Ginseng Cells

The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.

Pegylated interferon α-2b up-regulates specific CD8+ T cells in patients with chronic hepatitis B

AIM:To investigate the effect of pegylated interferon (IFN) α-2b on specific CD8+ T lymphocytes in patients with chronic hepatitis B (CHB). METHODS:Twenty-one patients with CHB were treated with pegylated IFN α-2b. Periphery blood mononuclear cells were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation (density:1.077 g/L,Pharmingen) at weeks 0,4,8,12,and 24,respectively. Frequency of circulating hepatitis B virus (HBV) epitope-specific CD8 T cells was detected by flow cytometry. Cytokines were detected by cytometric bead assay. RESULTS:The frequency of circulating HBV core or env-specific CD8 T cells was higher (P < 0.05),the number of HBV core specific CD8 T cells was greater at week 24 (P < 0.05),the level of Th1-type cytokines [interleukin (IL)-12,tumor necrosis factor-α,and IFN-γ] was higher,while that of Th2-type cytokines (IL-4,IL-6,and IL-10) was lower in responders than in nonresponders (P < 0.05) after pegylated IFN α-2b treatment. The IL-6 level was correlated with HBV DNA (r = 0.597,P = 0.04),while the inducible protein-10 (IP-10) level was correlated with serum alanine aminotransferase (ALT) (r = 0.545,P = 0.005). The IP-10 level at week 8 after pegylated IFN α-2b treatment could predict the normalization of ALT in CHB patients (positive predict value = 56%,negative predict value = 92%). CONCLUSION:Pegylated IFN α-2b can enhance the immune response of CHB patients by increasing the frequency of HBV specific CD8+ T cells and regulating the Th1/Th2 cytokines.

Study on pharmacodynamics of recombinant interferon α-2b suppository

Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the antiviral activity of this drug as well as yingtelong and axiluowei as positive control.The guinea pig model of vaginitis and skin infection caused by HSV-2 infection were established,treated with IFNα-2b suppository at dosages of 60000、180000、540000 IU,using IFNα-2b injection 180000 IU·kg-1 as controls.Score the pathological changes of appearance and skin,the virus activities of vaginal secretion and tissue sections of viginae were assayed after treatment.Results The TD50 of IFN α-2b and yingtelong for Vero cells was(>100)μg·mL-1 and(>100000)IU·mL-1,respectively.The IC50 of IFN α-2b and yingtelong and axiluowei for Herpes virus type 1 was(0.29±0.08)μg·mL-1 and(185.0±28.8)IU·mL-1 and(0.19±0.03)μg·mL-1,respectively.The mean scores for vaginal and skin lesion of the treated groups were lower than those of untreated group.Among these concentrations,the IFNα-2b suppository of 540000 IU·kg-1 group.Showed highest anti-viral activity.The virus activity in vaginal secretion of treated group was lower than that of untreated group too(P<0.01 or P<0.05).Tissue sections of viginae after treatment with IFNα-2b suppository showed significantly therapeutical effects on the degrees of vaginal lesion.At the same dosage,The anti-HSV activity of IFNα-2b suppository was also compared with IFNα-2b injection,the results showed that the activity of suppository of 540000 IU·kg-1 group was similar to that of the injection.Conclusions The IFNα-2b suppository has anti-viruses function both in vivo and in vitro.

IN VITRO CO-STIMULATORY ACTIVITY OF HUMAN B7.2(IgV+C) PROTEIN PRODUCED BY ENGINEERED BACTERIA

Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

Effects of interferon α-2b on liver function, complement level and oxidative stress in patients with hepatitis B

Objective:To observe the effect of interferonα-2b treatment on liver function,liver fibrosis,complement protein and oxidative stress in patients with hepatitis B.Methods:A total of 100 patients with hepatitis B in our hospital were randomly divided into the control group and the observation group,with 50 cases in each group.After admission,patients in the control group were treated with entecavir,while patients in the observation group were treated with interferonα-2b combined with entecavir.The levels of serum total bilirubin(TBil),aspartate aminotransferase(AST),alanine aminotransferase(ALT),type III procollagen(PCIII),type IV collagen(CIV),complement C3 protein(C3),complement C4 protein(C4),malondialdehyde(MDA),superoxide dismutase(SOD)and nitric oxide(NO)were compared between the two groups before and after treatment.Results:After treatment,the levels of TBil,AST,ALT,PCIII,CIV,MDA and NO in serum of patients with hepatitis B in both groups were significantly lower than those before treatment,and the levels of C3,C4 and SOD were significantly higher than those before treatment(P<0.05).After treatment,the levels of TBil,AST,ALT,PC III,C IV,MDA and NO in serum of patients in the observation group were significantly lower than those in the control group,while the levels of C3,C4 and SOD in serum of patients in the observation group were significantly higher than those in the control group(P<0.05).Conclusions:The combination of interferonα-2b and entecavir has a good curative effect on hepatitis B.It can significantly improve the liver function and immune function of patients,delay the process of liver fibrosis and reduce oxidative stress injury.It is worthy of clinical promotion.

SYNERGIC CYTOTOXICITY TO GASTRIC CANCER CELLS BY COMBINED USE OF TRICHOSANTHIN ANDRECOMBINANT INTERFERON α-2B

Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of TCS, rIFN α- 2b and their combination against digestive system cancer cell SGC- 7901.Results In the experiment in vitro, TCS, rIFN α- 2b both had direct, dose dependent cytotoxicity againstSGC - 7901. Their combined use demonstrated a toxicity signijicantly higher than that of the two drugs used alone,showing a signilicant synergic effect. This synergic cytotoxicity was confirmed in the animal experiment.Conclusion Combined use of TCS and rIFN α - 2b decreases the therapeutic dose of TCS and its toxic adverseellect, and this synergic effect is favorable to the clinical use of TCS protein against gastric cancer.

Effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with CINⅢ complicated by high-risk HPV positive

Objective: To study the effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with cervical intraepithelial neoplasia (CIN) Ⅲ complicated by high-risk HPV positive. Methods: Patients who were diagnosed with CINⅢ and high-risk HPV positive and underwent conization in the 3201 Hospital Affiliated to Xi'an Jiaotong University between June 2014 and February 2017 were selected and randomly divided into the observation group who received preoperative anti-HPV bioprotein dressing combined with interferon α-2b therapy and the control group who received no special treatment. CIN lesion was collected to determine the expression of pro-proliferation molecules, pro-apoptosis molecules and epithelial-mesenchymal transition molecules. Results: Rsf1, Piwil2, TOPK, p38MAPK, ERK, Snail, Twist, N-cadherin and Vimentin mRNA expression in cervical intraepithelial neoplasia lesions of observation group were greatly lower than those of control group whereas LRIG3, SARI, IEX-1, FHIT and E-cadherin mRNA expression were greatly higher than those of control group. Conclusion: Anti-HPV bioprotein dressing combined with interferon α-2b therapy can inhibit the proliferation and invasive growth of tumor cells in patients with CINⅢ complicated by high-risk HPV positive.

人乳头瘤病毒感染对乳腺癌患者p53、bcl-2和c-erbB-2表达及预后的影响

目的探讨人乳头瘤病毒(HPV)感染对乳腺癌患者p53、B淋巴细胞瘤2基因(bcl-2)和人类表皮生长因子受体2(c-erbB-2)的表达及预后的影响。方法选取2017年2月至2020年1月在首都医科大学附属北京友谊医院行乳腺癌根治术的乳腺癌患150例,原位杂交法检测乳腺癌患者HPV感染情况,按照是否有HPV感染将150例患者分成HPV感染组86例和未感染组64例。采用免疫组化法检测乳腺癌组织中p53、bcl-2和c-erbB-2蛋白表达情况。收集患者的临床特征并分析HPV感染及p53、bcl-2、c-erbB-2蛋白阳性表达与临床特征的关系;随访患者术后2年的生存情况。结果150例乳腺癌患者中HPV感染86例,感染率57.33%。HPV感染组p53、c-erbB-2阳性表达率高于未感染组,bcl-2阳性表达率低于未感染组(P<0.05);HPV感染与国际妇产科协会(FIGO)肿瘤分期有关,p53阳性表达淋巴转移有关,bcl-2阳性表达与FIGO分期有关,c-erbB-2阳性表达与组织学分级、淋巴转移有关(P<0.05)。HPV感染组2年生存率低于未感染组[82.55%(71/86)vs.93.75%(60/64),χ2=4.155,P=0.042]。结论乳腺癌患者HPV感染率较高,HPV感染可能通过促进c-erbB-2和p53表达以及抑制bcl-2表达,促进乳腺癌进展并导致患者预后不良。

人干扰素α-2b基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b(Human interferon α-2b,HuIFN α-2b)编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。

清热解毒方联合干扰素a-2b治疗宫颈高危型人乳头瘤病毒持续感染随机平行对照研究

[目的]观察清热解毒方联合干扰素a-2b治疗宫颈高危型人乳头瘤病毒(HR-HPV)持续感染疗效。[方法]使用随机平行对照方法,将68例住院患者按随机数字表法分为两组。对照组72例干扰素a-2b,隔日一粒,睡前放置于阴道后穹隆接近宫颈口处。治疗组142例清热解毒方(山药、车前子、苍术、鸡冠花、猪苓各15g,白花蛇舌草、生薏苡仁、蒲公英各30g,柴胡、芡实各10g,炒白术20g),1剂/d,水煎300mL,早晚口服,月经干净后服用,经期停药。西药治疗同对照组。连续治疗30d为1疗程。观测临床症状、血液学检查、HPV复查、不良反应。连续治疗3疗程,判定疗效。[结果]治疗组痊愈93例,有效34例,无效15例,总有效率89.44%;对照组痊愈21例,有效39例,无效12例,总有效率83.33%。转阴93例,转阴率65.49%。临床疗效治疗组优于对照组(P<0.05)。治疗组HR-HPV病毒转阴率优于对照组(P<0.01)。[结论]清热解毒方联合干扰素a-2b治疗HR-HPV持续感染效果显著,值得推广使用。

按大肠杆菌高表达序列设计的人干扰素α-2b基因的化学合成和克隆

根据大肠杆菌高表达序列重新设计了人干扰素α-2b基因编码区的核苷酸序列,应用化学合成的方法合成了多个互补DNA片段,经互补片段分别退火和连接后,将完整编码区分两段分别克隆于pUC19质粒中,进行DNA序列分析,分别选取序列完全正确的两种克隆片段进行重组.获得了与设计序列完全一致的含有完整编码区的人干扰素α-2b基因.

人重组干扰素-α-2b治疗子宫内膜异位症的临床分析

目的观察分析人重组干扰素-α-2b治疗子宫内膜异位症的临床疗效。方法选取2010年6月-2012年6月子宫内膜异位症的患者56例,按照随机数字法分成观察组和对照组,各28例,观察组使用人重组干扰素-α-2b治疗,对照组使用戈舍瑞林治疗,观察对比两组治疗效果。结果两组术后血清CA125、EMAb水平术后1、2、3个月与术前对比存在明显差异(P<0.05),具有统计学意义;两组治疗期间药物不良反应发生率进行比较存在一定差异(P<0.05),具有统计学意义。结论人重组干扰素-α-2b治疗子宫内膜异位症的临床疗效显著,与常规使用戈舍瑞林治疗作用类似,药物不良反应表现在使用早期较多,但持续时间短,停药后皆能自行消失,安全性较高。

重组人干扰素α-2b凝胶联合微波治疗慢性宫颈炎临床体会

目的探讨重组人干扰素α-2 b凝胶联合微波治疗慢性宫颈炎的疗效。方法按照数字随机表法将我院妇科门诊收治的282例慢性宫颈炎患者分为微波治疗组(对照组)和重组人干扰素α-2 b凝胶联合微波治疗组(观察组),每组各141例,比较两组患者的临床疗效。结果观察组患者治疗总有效率为94.3%高于对照组患者的85.1%,对比两组患者在治疗总有效率,P<0.05,差异具有统计学意义。结论重组人干扰素α-2 b凝胶联合微波治疗慢性宫颈炎的疗效较为理想,是一种理想的治疗方案。

Induction of anti-hepatoma immunity by recombinant retrovirus expressing B7-1 /B7-2 costimulatory molecules

Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.

Development of novel interferon alpha2b muteins and study the pharmacokinetic and biodistribution profiles in animal model

Novel human interferon alpha 2b (hIFNα2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic profile. In addition, the influence of charge on the protein structure was tested in vivo for the biodistribution pattern. Codon substitutions were performed by Polymerase Chain Reaction (PCR)-based site-directed mutagenesis on a previously constructed synthetic hIFNα2b open reading frame (ORF) cloned in pET32b expression plasmid. The result of nucleotide sequencing analysis confirmed that all codons were replaced successfully without any additional mutation. Three mutant forms of hIFNα2b ORF were overexpressed in Escherichia coli BL21 (DE3) resulted in three muteins: hIFNα2b C2D, hIFNα2b C99D, hIFNα2b C2D C99D. To follow the kinetic and localization of the mutein interferon after intravenous administration, Tc99m was used to label the proteins. In particular of elimination half-life, it was shown that hIFNα2b C2D C99D > hIFNα2bC2D > hIFNα2bC99D > wild type. hIFNα2b C2D C99D mutein showed highest blood accumulation after 30 minutes administration. Taken together, the charge of hIFNα2b seems to be responsible for the fate of hIFNα2b in vivo.

端粒酶、人类表皮生长因子受体2和c-myc基因在宫颈病变中的表达及与高危型人乳头瘤病毒感染的相关性

目的研究端粒酶基因(hTERC)、人类表皮生长因子受体2(HER-2)基因和c-myc基因在不同程度宫颈病变的表达情况及与高危型人乳头瘤病毒(HPV)感染的相关性。方法选取236例就诊或筛查患者,行TCT、HPV、hTERC、HER-2和c-myc基因检测。对任一阳性患者行阴道镜下多点活检,进行病理检测。结果 1236例中有108例进行宫颈活检,在正常/慢性宫颈炎、CINⅠ、CINⅡ-Ⅲ及浸润性子宫颈癌中hTERC阳性表达率分别为16.0%、39.5%、64.5%、100.0%;HER-2阳性表达率分别为4.0%、20.9%、45.2%、100.0%;c-myc阳性表达率分别为8.0%、30.2%、54.8%、100.0%;HPV阳性率分别为40.0%、67.4%、80.6%、100.0%。2不同病理结果之间hTERC、HER-2和c-myc阳性率比较差异均有统计学意义(P<0.05),随着病理结果的严重程度,hTERC、HER-2和c-myc的阳性率逐渐增加(P<0.01)。3随着宫颈病变程度的加深,HPV感染者中hTERC、HER-2和c-myc基因的阳性扩增率也增高(P<0.05)。结论hTERC、HER-2、c-myc基因的阳性表达率与宫颈病变程度、高危型HPV感染密切相关,在宫颈癌的发生发展中可能起着重要作用。

NLRP3、AIM2、IFI16炎症小体在慢性乙型病毒性肝炎患者PBMC中的活化水平和与HBV感染的相关性分析

目的:探讨乙肝病毒(HBV)是否激活了慢性乙型病毒性肝炎(CHB)患者外周血单个核细胞(PBMCs)内核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、黑色素瘤缺乏因子2(AIM2)和干扰素诱导蛋白16(IFI16)炎症小体,分析HBV影响炎症小体活化的可能机制.方法:收集感染内科临床确诊CHB患者35例.同时选取健康住院医师28例为对照.以常规淋巴细胞分层液密度梯度离心法分离健康对照组和CHB患者组静脉血得到PBMCs,采用逆转录、实时荧光定量PCR检测CHB患者组和健康对照组PBMCs NLRP3、AIM2、IFI16、凋亡相关的斑点样蛋白(ASC)、半胱天冬酶1(CASP1)、IL-1β、IL-18 mRNA表达水平,ELISA法检测两组血清中IL-1β蛋白分泌水平.结果:CHB患者组和健康对照组PBMCs ASC、NLRP3、AIM2、IL-1β、IL-18 mRNA表达水平及两组血清IL-1β蛋白分泌水平无显著性差异.CHB患者组PBMCs IFI16、CASP1 mRNA表达水平显著上调,且IFI16 mRNA表达水平与患者血清HBV DNA载量显著正相关(r=0.699 8,P<0.01).结论:慢性HBV感染未导致CHB患者PBMCs NLRP3、AIM2炎症小体的活化;尽管HBV DNA可能诱导了CHB患者IFI16炎症小体的高表达,但通过抑制pro-caspase-1的活化、IL-1β的表达,HBV阻断了IFI16炎症小体的活化效应.