流感黏膜疫苗的初步研究
发布时间：2018-09-11 06:36
【摘要】： 流感病毒主要感染呼吸道上皮细胞,通过呼吸道黏膜进入动物和人体。因此,黏膜免疫在预防流感病毒感染的研究领域中显得尤为重要。 本研究在鸡胚中进行流感病毒的培养,收获病毒后进行灭活、浓缩及纯化得到流感全病毒灭活抗原,经裂解后得到流感病毒裂解抗原。将全病毒灭活抗原分别与霍乱毒素B亚单位(Cholera toxin B, CTB)、蛋白体(Proteosomes)、氢氧化铝(Al(OH)3)三种佐剂配伍,将裂解病毒分别与上述三种佐剂配伍,进一步探讨其滴鼻免疫小鼠和豚鼠后诱导的免疫效果,最后通过比较筛选出最有效的流感黏膜疫苗。 1.本试验从流感病毒的培养,浓缩,纯化等方面优化了制备流感全病毒灭活抗原的条件。试验采用鸡胚培养流感病毒,A型病毒在33℃孵箱中培养56 h,B型病毒在33℃孵箱中培养72 h,收获病毒后,用终浓度为0.02%的甲醛4℃灭活12 h,采用截流分子量为10 ku的超滤膜包进行流感病毒的浓缩,浓缩倍数大约控制在25～30倍之间,之后采用非连续蔗糖密度梯度液(从离心管底向上依次为60%,45%,30%的蔗糖溶液), 4℃,30000 r/min,离心2 h,纯化浓缩的病毒液,透析去糖,PEG浓缩后得到最终的单价灭活病毒液。流感病毒裂解抗原制备方法基本同流感全病毒灭活抗原。我们所采用的裂解剂是终浓度为5%的Triton X-100,20℃,裂解12 h。经过灭活验证试验,单向免疫扩散试验、电镜分析等一系列鉴定,用该套方法制备的流感全病毒灭活抗原可用于后续试验。 2.在流感全病毒抗原组中,单独以流感全病毒抗原滴鼻免疫小鼠,其sIgA以及HAI水平均高于各佐剂组,说明,单独以流感全病毒抗原滴鼻免疫小鼠,不但刺激机体产生体液免疫应答,且诱导了机体的黏膜免疫应答;而在流感裂解病毒抗原组中,以蛋白体为佐剂的流感裂解抗原滴鼻免疫小鼠,引起的特异性血清IgG、IgA、IgM及HAI明显高于流感裂解抗原单独滴鼻组及其余佐剂组,说明以蛋白体作为佐剂可显著提高抗原的免疫原性,刺激机体产生较强的体液免疫应答,而且诱导呼吸道黏膜产生sIgA,加强了机体的局部黏膜免疫应答,这对于流感病毒的抗感染免疫具有关键作用。 3.用不同剂量的蛋白体-三价流感裂解抗原和三价流感裂解抗原滴鼻免疫小鼠,结果显示:佐剂组中,当免疫剂量达到4μg/只时,可显著提高小鼠血清IgG,IgM以及HAI水平,小鼠脾脏T淋巴细胞CD4+/CD8+值显著高于其余各试验组,说明,该剂量组机体体液免疫反应强于其余各剂量组,且不引起免疫抑制;佐剂组中,当免疫剂量达到4μg/只时,呼吸道黏膜抗体sIgA水平明显升高,说明,该剂量组黏膜免疫反应明显高于其余各剂量组;无佐剂组,各剂量组各项检测指标均处于较低水平,说明,单独以三价流感裂解抗原滴鼻免疫小鼠,仅能刺激机体产生微弱的免疫反应;在相同剂量条件下,佐剂组各项检测指标均显著高于无佐剂组,说明,蛋白体作为流感裂解抗原的佐剂滴鼻免疫小鼠,可明显提高机体的系统免疫应答和黏膜免疫反应。 4.以不同剂量三价流感全病毒抗原滴鼻免疫小鼠,在一定抗原剂量范围内,血清IgG、HAI以及sIgA呈现抗原剂量依赖性,即随抗原剂量的加大,各项检测指标水平升高,当免疫剂量达到8μg/只时,血清IgG、HAI以及sIgA达到峰值,抗原剂量再次加大时,各检测指标出现下降,说明8μg/只剂量组可明显提高机体系统免疫应答和呼吸道黏膜免疫应答。而小鼠脾脏T淋巴细胞CD4+/CD8+值随抗原剂量的加大而降低,当免疫剂量达到8μg/只时,CD4+/CD8+值达到最低值,再次提高抗原剂量时,该值回升,说明,8μg/只剂量组可明显提高机体的细胞免疫应答。以不同剂量三价流感全病毒抗原滴鼻免疫豚鼠,6μg/只剂量组各项指标明显低于10μg/只和20μg/只剂量组,而10μg/只和20μg/只剂量组各项检测指标水平相当,在免疫应答反应相当的情况下,较低剂量为最佳剂量,因此10μg/只为最佳剂量。
[Abstract]:Influenza viruses mainly infect the epithelial cells of the respiratory tract and enter animals and humans through the mucosa of the respiratory tract.
Influenza viruses were cultured in chicken embryos, inactivated after harvesting, concentrated and purified to obtain inactivated antigens of influenza viruses. The inactivated antigens of influenza viruses were divided into three subunits, Cholera toxin B (CTB), proteosomes and aluminum hydroxide (Al (OH) 3. Compatibility of adjuvants, lysis virus and the above three adjuvants were compatible to further explore the nasal immunization of mice and guinea pigs induced immune effect, and finally through comparison screened out the most effective influenza mucosal vaccine.
1. The conditions of preparing inactivated antigen of influenza virus were optimized from the aspects of culture, concentration and purification of influenza virus. Influenza virus was cultured in chicken embryo, A virus was cultured in incubator at 33 C for 56 hours, B virus was cultured in incubator at 33 C for 72 hours. After harvesting the virus, formaldehyde with the final concentration of 0.02% was inactivated at 4 C for 12 hours, and cut off fraction was used. Ultrafiltration packages with 10 Ku were used to concentrate influenza virus. The concentrating multiple was controlled between 25 and 30 times. Then discontinuous sucrose density gradient solution (60%, 45% and 30% sucrose solution from the bottom to the top of centrifugal tube) was used to purify the concentrated virus at 4 C, 30 000 r/min, and centrifuged for 2 hours. After dialysis, the final single was obtained by PEG concentration. Influenza virus lysis antigen preparation method is basically the same as the whole influenza virus inactivated antigen. We used the final concentration of 5% Triton X-100,20 C, cleavage 12 h. After inactivation validation test, one-way immunodiffusion test, electron microscopic analysis and a series of identification, using this set of methods to prepare inactivated influenza virus antibody. It can be used for subsequent experiments.
2. The levels of sIgA and HAI in mice inoculated with influenza virus antigen by nose dropping were higher than those in adjuvant groups, indicating that the mice inoculated with influenza virus antigen by nose dropping not only stimulated humoral immune response, but also induced mucosal immune response in mice inoculated with influenza virus antigen. The specific serum IgG, IgA, IgM and HAI induced by intranasal immunization of mice with influenza lysis antigen with proteasome as adjuvant were significantly higher than those induced by intranasal drip of influenza lysis antigen alone and other adjuvant groups, indicating that proteasome as adjuvant could significantly improve the immunogenicity of the antigen, stimulate the body to produce a strong humoral immune response and induce expiration. The production of sIgA in the airway mucosa strengthens the local mucosal immune response, which plays a key role in the anti-infective immunity of influenza virus.
3. Different doses of proteasome-trivalent influenza lysis antigen and trivalent influenza lysis antigen were used to immunize mice nasally. The results showed that the levels of serum IgG, IgM and HAI were significantly increased in the adjuvant group when the immune dosage reached 4 ug/mouse. The CD4+/CD8+ values of spleen T lymphocyte in the mice were significantly higher than those in the other experimental groups. The humoral immune response was stronger than that of the other dosage groups and did not induce immunosuppression; in the adjuvant group, when the immune dose reached 4 ug/kg, the level of respiratory mucosal antibody sIgA increased significantly, indicating that the mucosal immune response of the dose group was significantly higher than that of the other dosage groups; in the non-adjuvant group, the detection indexes of each dose group were at a lower level, said the adjuvant group. The results showed that intranasal immunization of mice with trivalent influenza lysis antigen alone could only stimulate the body to produce weak immune response; at the same dosage, the detection indexes of adjuvant group were significantly higher than those of non-adjuvant group, indicating that the proteasome as adjuvant of influenza lysis antigen could significantly improve the systemic immune response and viscosity of the body. Membrane immunoreaction.
4. The mice were immunized with different doses of trivalent influenza virus antigen by nasal dropping. In a certain dose range, serum IgG, HAI and sIgA showed dose dependence, that is, with the increase of antigen dosage, the levels of each detection index increased. When the immune dosage reached 8 UG per mouse, serum IgG, HAI and sIgA reached the peak value, and the antigen dosage increased again. The CD4 + / CD8 + value of spleen T lymphocyte in mice decreased with the increase of antigen dosage. When the immune dosage reached 8 UG / only, the CD4 + / CD8 + value reached the lowest value, and when the antigen dosage was increased again, the value returned to the lowest value. When guinea pigs were intranasally immunized with different doses of trivalent influenza virus antigen, the indexes in the 6-ug/only dosage group were significantly lower than those in the 10-ug/only and 20-ug/only dosage groups, while those in the 10-ug/only and 20-ug/only dosage groups were comparable, and the immune response was comparable. In the case, the lower dose is the best dose, so 10 g/ is the best dose.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
[Abstract]:Influenza viruses mainly infect the epithelial cells of the respiratory tract and enter animals and humans through the mucosa of the respiratory tract.
Influenza viruses were cultured in chicken embryos, inactivated after harvesting, concentrated and purified to obtain inactivated antigens of influenza viruses. The inactivated antigens of influenza viruses were divided into three subunits, Cholera toxin B (CTB), proteosomes and aluminum hydroxide (Al (OH) 3. Compatibility of adjuvants, lysis virus and the above three adjuvants were compatible to further explore the nasal immunization of mice and guinea pigs induced immune effect, and finally through comparison screened out the most effective influenza mucosal vaccine.
1. The conditions of preparing inactivated antigen of influenza virus were optimized from the aspects of culture, concentration and purification of influenza virus. Influenza virus was cultured in chicken embryo, A virus was cultured in incubator at 33 C for 56 hours, B virus was cultured in incubator at 33 C for 72 hours. After harvesting the virus, formaldehyde with the final concentration of 0.02% was inactivated at 4 C for 12 hours, and cut off fraction was used. Ultrafiltration packages with 10 Ku were used to concentrate influenza virus. The concentrating multiple was controlled between 25 and 30 times. Then discontinuous sucrose density gradient solution (60%, 45% and 30% sucrose solution from the bottom to the top of centrifugal tube) was used to purify the concentrated virus at 4 C, 30 000 r/min, and centrifuged for 2 hours. After dialysis, the final single was obtained by PEG concentration. Influenza virus lysis antigen preparation method is basically the same as the whole influenza virus inactivated antigen. We used the final concentration of 5% Triton X-100,20 C, cleavage 12 h. After inactivation validation test, one-way immunodiffusion test, electron microscopic analysis and a series of identification, using this set of methods to prepare inactivated influenza virus antibody. It can be used for subsequent experiments.
2. The levels of sIgA and HAI in mice inoculated with influenza virus antigen by nose dropping were higher than those in adjuvant groups, indicating that the mice inoculated with influenza virus antigen by nose dropping not only stimulated humoral immune response, but also induced mucosal immune response in mice inoculated with influenza virus antigen. The specific serum IgG, IgA, IgM and HAI induced by intranasal immunization of mice with influenza lysis antigen with proteasome as adjuvant were significantly higher than those induced by intranasal drip of influenza lysis antigen alone and other adjuvant groups, indicating that proteasome as adjuvant could significantly improve the immunogenicity of the antigen, stimulate the body to produce a strong humoral immune response and induce expiration. The production of sIgA in the airway mucosa strengthens the local mucosal immune response, which plays a key role in the anti-infective immunity of influenza virus.
3. Different doses of proteasome-trivalent influenza lysis antigen and trivalent influenza lysis antigen were used to immunize mice nasally. The results showed that the levels of serum IgG, IgM and HAI were significantly increased in the adjuvant group when the immune dosage reached 4 ug/mouse. The CD4+/CD8+ values of spleen T lymphocyte in the mice were significantly higher than those in the other experimental groups. The humoral immune response was stronger than that of the other dosage groups and did not induce immunosuppression; in the adjuvant group, when the immune dose reached 4 ug/kg, the level of respiratory mucosal antibody sIgA increased significantly, indicating that the mucosal immune response of the dose group was significantly higher than that of the other dosage groups; in the non-adjuvant group, the detection indexes of each dose group were at a lower level, said the adjuvant group. The results showed that intranasal immunization of mice with trivalent influenza lysis antigen alone could only stimulate the body to produce weak immune response; at the same dosage, the detection indexes of adjuvant group were significantly higher than those of non-adjuvant group, indicating that the proteasome as adjuvant of influenza lysis antigen could significantly improve the systemic immune response and viscosity of the body. Membrane immunoreaction.
4. The mice were immunized with different doses of trivalent influenza virus antigen by nasal dropping. In a certain dose range, serum IgG, HAI and sIgA showed dose dependence, that is, with the increase of antigen dosage, the levels of each detection index increased. When the immune dosage reached 8 UG per mouse, serum IgG, HAI and sIgA reached the peak value, and the antigen dosage increased again. The CD4 + / CD8 + value of spleen T lymphocyte in mice decreased with the increase of antigen dosage. When the immune dosage reached 8 UG / only, the CD4 + / CD8 + value reached the lowest value, and when the antigen dosage was increased again, the value returned to the lowest value. When guinea pigs were intranasally immunized with different doses of trivalent influenza virus antigen, the indexes in the 6-ug/only dosage group were significantly lower than those in the 10-ug/only and 20-ug/only dosage groups, while those in the 10-ug/only and 20-ug/only dosage groups were comparable, and the immune response was comparable. In the case, the lower dose is the best dose, so 10 g/ is the best dose.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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