流感黏膜疫苗的初步研究
發(fā)布時間：2018-09-11 06:36
【摘要】： 流感病毒主要感染呼吸道上皮細(xì)胞,通過呼吸道黏膜進(jìn)入動物和人體。因此,黏膜免疫在預(yù)防流感病毒感染的研究領(lǐng)域中顯得尤為重要。 本研究在雞胚中進(jìn)行流感病毒的培養(yǎng),收獲病毒后進(jìn)行滅活、濃縮及純化得到流感全病毒滅活抗原,經(jīng)裂解后得到流感病毒裂解抗原。將全病毒滅活抗原分別與霍亂毒素B亞單位(Cholera toxin B, CTB)、蛋白體(Proteosomes)、氫氧化鋁(Al(OH)3)三種佐劑配伍,將裂解病毒分別與上述三種佐劑配伍,進(jìn)一步探討其滴鼻免疫小鼠和豚鼠后誘導(dǎo)的免疫效果,最后通過比較篩選出最有效的流感黏膜疫苗。 1.本試驗從流感病毒的培養(yǎng),濃縮,純化等方面優(yōu)化了制備流感全病毒滅活抗原的條件。試驗采用雞胚培養(yǎng)流感病毒,A型病毒在33℃孵箱中培養(yǎng)56 h,B型病毒在33℃孵箱中培養(yǎng)72 h,收獲病毒后,用終濃度為0.02%的甲醛4℃滅活12 h,采用截流分子量為10 ku的超濾膜包進(jìn)行流感病毒的濃縮,濃縮倍數(shù)大約控制在25～30倍之間,之后采用非連續(xù)蔗糖密度梯度液(從離心管底向上依次為60%,45%,30%的蔗糖溶液), 4℃,30000 r/min,離心2 h,純化濃縮的病毒液,透析去糖,PEG濃縮后得到最終的單價滅活病毒液。流感病毒裂解抗原制備方法基本同流感全病毒滅活抗原。我們所采用的裂解劑是終濃度為5%的Triton X-100,20℃,裂解12 h。經(jīng)過滅活驗證試驗,單向免疫擴(kuò)散試驗、電鏡分析等一系列鑒定,用該套方法制備的流感全病毒滅活抗原可用于后續(xù)試驗。 2.在流感全病毒抗原組中,單獨(dú)以流感全病毒抗原滴鼻免疫小鼠,其sIgA以及HAI水平均高于各佐劑組,說明,單獨(dú)以流感全病毒抗原滴鼻免疫小鼠,不但刺激機(jī)體產(chǎn)生體液免疫應(yīng)答,且誘導(dǎo)了機(jī)體的黏膜免疫應(yīng)答;而在流感裂解病毒抗原組中,以蛋白體為佐劑的流感裂解抗原滴鼻免疫小鼠,引起的特異性血清IgG、IgA、IgM及HAI明顯高于流感裂解抗原單獨(dú)滴鼻組及其余佐劑組,說明以蛋白體作為佐劑可顯著提高抗原的免疫原性,刺激機(jī)體產(chǎn)生較強(qiáng)的體液免疫應(yīng)答,而且誘導(dǎo)呼吸道黏膜產(chǎn)生sIgA,加強(qiáng)了機(jī)體的局部黏膜免疫應(yīng)答,這對于流感病毒的抗感染免疫具有關(guān)鍵作用。 3.用不同劑量的蛋白體-三價流感裂解抗原和三價流感裂解抗原滴鼻免疫小鼠,結(jié)果顯示:佐劑組中,當(dāng)免疫劑量達(dá)到4μg/只時,可顯著提高小鼠血清IgG,IgM以及HAI水平,小鼠脾臟T淋巴細(xì)胞CD4+/CD8+值顯著高于其余各試驗組,說明,該劑量組機(jī)體體液免疫反應(yīng)強(qiáng)于其余各劑量組,且不引起免疫抑制;佐劑組中,當(dāng)免疫劑量達(dá)到4μg/只時,呼吸道黏膜抗體sIgA水平明顯升高,說明,該劑量組黏膜免疫反應(yīng)明顯高于其余各劑量組;無佐劑組,各劑量組各項檢測指標(biāo)均處于較低水平,說明,單獨(dú)以三價流感裂解抗原滴鼻免疫小鼠,僅能刺激機(jī)體產(chǎn)生微弱的免疫反應(yīng);在相同劑量條件下,佐劑組各項檢測指標(biāo)均顯著高于無佐劑組,說明,蛋白體作為流感裂解抗原的佐劑滴鼻免疫小鼠,可明顯提高機(jī)體的系統(tǒng)免疫應(yīng)答和黏膜免疫反應(yīng)。 4.以不同劑量三價流感全病毒抗原滴鼻免疫小鼠,在一定抗原劑量范圍內(nèi),血清IgG、HAI以及sIgA呈現(xiàn)抗原劑量依賴性,即隨抗原劑量的加大,各項檢測指標(biāo)水平升高,當(dāng)免疫劑量達(dá)到8μg/只時,血清IgG、HAI以及sIgA達(dá)到峰值,抗原劑量再次加大時,各檢測指標(biāo)出現(xiàn)下降,說明8μg/只劑量組可明顯提高機(jī)體系統(tǒng)免疫應(yīng)答和呼吸道黏膜免疫應(yīng)答。而小鼠脾臟T淋巴細(xì)胞CD4+/CD8+值隨抗原劑量的加大而降低,當(dāng)免疫劑量達(dá)到8μg/只時,CD4+/CD8+值達(dá)到最低值,再次提高抗原劑量時,該值回升,說明,8μg/只劑量組可明顯提高機(jī)體的細(xì)胞免疫應(yīng)答。以不同劑量三價流感全病毒抗原滴鼻免疫豚鼠,6μg/只劑量組各項指標(biāo)明顯低于10μg/只和20μg/只劑量組,而10μg/只和20μg/只劑量組各項檢測指標(biāo)水平相當(dāng),在免疫應(yīng)答反應(yīng)相當(dāng)?shù)那闆r下,較低劑量為最佳劑量,因此10μg/只為最佳劑量。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392
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