本文關(guān)鍵詞：滅活輪狀病毒與滅活甲肝病毒聯(lián)合疫苗的制備及免疫學效果評價　出處：《昆明醫(yī)學院》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 輪狀病毒 甲肝病毒 聯(lián)合疫苗 免疫反應(yīng)

【摘要】：使用聯(lián)合疫苗早在上世紀八十年代末,世界衛(wèi)生組織(WHO)在制定兒童疫苗免疫計劃(CVI)就提出能否用20-30年時間研制出更多的一次免疫預(yù)防多種疾病的疫苗。因此,研制聯(lián)合疫苗不僅是目前也是未來疫苗研發(fā)的方向,聯(lián)合疫苗不僅解決受接種者的經(jīng)濟負擔,也減輕了精神負擔。目前已有一些聯(lián)合疫苗用于我國的免疫規(guī)劃,包括百白破,麻腮風,甲乙肝疫苗等,但至今我國還沒有以輪狀病毒為基礎(chǔ)的聯(lián)合疫苗。 人輪狀病毒(human rotavirus, HRV)是世界范圍內(nèi)引起5歲以內(nèi)嬰幼兒嚴重腹瀉最主要的病原體,HRV感染是造成嬰幼兒腹瀉死亡的主要原因。至今每年約有60萬嬰兒死于RV感染引起的腹瀉。目前的減毒RV活疫苗在預(yù)防RV感染中發(fā)揮了一定作用,但疫苗引起的副反應(yīng)如外源因子污染及可能發(fā)生毒力返祖的問題,尚未完全得到解決。世界衛(wèi)生組織(world health organization, WHO)正積極推動不同形式輪狀病毒疫苗的研究與開發(fā)。滅活RV也是WHO提倡研制的RV疫苗之一。為開展聯(lián)合疫苗的研制,我們已完成了實驗室規(guī)模滅活RV疫苗的制備,為聯(lián)合疫苗的研制提供了條件。 病毒性肝炎是由多種肝炎病毒引起的以肝臟損害為主的全身性急性或持續(xù)性傳染病,在世界各地均有發(fā)病流行。在肝炎患者中30-50%屬于甲型肝炎。甲肝的流行,其發(fā)生與社會經(jīng)濟的發(fā)達程度,環(huán)境衛(wèi)生水平密切相關(guān)。發(fā)達國家感染率達到50-1000/10萬人,發(fā)展中國家為100-300/10萬人。我國大部分地區(qū)屬于甲肝高流行區(qū)。由于甲肝疫苗的使用,我國甲肝的流行與發(fā)病率已大幅下降。其中滅活甲肝疫苗發(fā)揮了重要的作用。 如果能將兩種或兩種以上的有效疫苗配制成聯(lián)合疫苗將減輕受種者的經(jīng)濟和精神負擔�；谶@一考慮,本研究認為IRV和IHAV疫苗理化性質(zhì)相同,免疫對象相同,免疫程序基本一致。因此,進行了滅活輪狀病毒與滅活甲肝病毒聯(lián)合疫苗的制備及免疫學效果評價。 實驗第一部分：聯(lián)合疫苗的制備。 實驗方法：規(guī)�；囵B(yǎng)的P[2]G3輪狀病毒收獲液,經(jīng)離心、微濾、超濾和層析等步驟純化,獲得結(jié)構(gòu)完整,呈典型的輪狀病毒特征,具有感染性,基因組未發(fā)生突變的病毒液,加入終濃度0.00925%甲醛進行滅活,先在56℃加熱30min,然后在37℃持續(xù)孵育至72h,經(jīng)盲傳三代證明沒有殘余毒力,病毒被完全滅活。經(jīng)抗原定量檢測,再將滅活的P[2]G3輪狀病毒與氫氧化鋁佐劑吸附,與成品甲肝試劑混合制備成聯(lián)合疫苗。 實驗第二部分：免疫學效果評價。 實驗方法：將小鼠分為空白組,對照組,實驗組。然后在小鼠上進行聯(lián)合疫苗的免疫效果評價。聯(lián)合疫苗經(jīng)肌肉注射,每次免疫間隔兩周,共免疫3次。自第1次免疫后兩周檢測小鼠血清中抗RV特異性抗體和糞便中的分泌性抗體,分析免疫后CD4+T淋巴細胞和CD8+T淋巴細胞的變化情況。 實驗結(jié)果：初次免疫后第2周,小鼠全部抗RV抗體陽轉(zhuǎn)；二次免疫后第4周時,檢測到抗HAV抗體；在第3次免疫后第6周時,抗體水平到達高峰小鼠血清抗RV酶標抗體滴度達80865.76±1953.97,抗RV分泌性抗體滴度為4.54±0.58,抗RV中和抗體效價為10240,抗甲肝抗體效價為28.50876±6.53。細胞免疫反應(yīng)CD4+T達到14.52±3.83,CD8+T達到3.9±0.72。經(jīng)t檢驗,P0.05。以上實驗結(jié)果表明,滅活輪狀病毒與滅活甲肝病毒聯(lián)合疫苗可誘導(dǎo)小鼠產(chǎn)生特異性抗RV體液免疫反應(yīng)和抗HAV體液免疫反應(yīng),也能產(chǎn)生明顯的細胞免疫反應(yīng)。這項研究為研制IRV和IHAV聯(lián)合疫苗提供了一些科學依據(jù)。
[Abstract]:The use of combined vaccine as early as in the last century at the end of 80s, WHO (WHO) in the development of vaccine immunization for children plan (CVI) proposed to 20-30 years in developing a vaccine immune prevention of various diseases more. Therefore, development of combined vaccine is not only the present also is the future direction of vaccine development, not only solved by combined vaccine with the economic burden, but also reduce the mental burden. There are some combined vaccine for China's immunization program, including DPT, MMR, a hepatitis B vaccine, but so far our country has not combined vaccine with rotavirus based.
Human rotavirus (human rotavirus HRV) is less than 5 year of severe diarrhea in infants and young children the main pathogen worldwide, HRV infection is the main cause of infantile diarrhea death. Every year about 600 thousand babies died of diarrhea caused by RV infection. The attenuated live vaccine RV play a role in the prevention of RV the infection, but the side effects caused by exogenous factors such as vaccine contamination and possible virulence reversion problems have not been completely solved. The WHO (World Health Organization, WHO) is actively promoting the research and development of different forms of rotavirus vaccine. Inactivated RV vaccine is one of the RV WHO to promote the development. For the development of joint the vaccine, we have completed the laboratory scale preparation of inactivated RV vaccine, provided the conditions for the development of vaccine.
Viral hepatitis is caused by a variety of viral hepatitis with liver damage mainly systemic acute or persistent infectious disease incidence in the popular all over the world. In patients with hepatitis 30-50% hepatitis. Hepatitis A epidemic belongs to, the development degree of social economy, is closely related to the level of environmental sanitation. The infection rate of developed countries 50-1000/10 million, 100-300/10 million people in developing countries. Most areas of our country belong to a high endemic area. Due to the use of hepatitis A vaccine, epidemic and the incidence of hepatitis A in China have dropped significantly. The inactivated hepatitis A vaccine has played an important role.
If two or more than two kinds of effective vaccine prepared combined vaccine will reduce the sort of economic and mental burden. Based on this consideration, this study shows that the IRV IHAV vaccine and physicochemical properties of the same object are the same, the immune, immune procedure is basically the same. Therefore, the preparation and evaluation of immune effect of inactivated live rotavirus and inactivated hepatitis A virus vaccine.
The first part of the experiment: the preparation of the combined vaccine.
Methods: P[2]G3 rotavirus harvest scale culture liquid by centrifugation, microfiltration, ultrafiltration and chromatography purification step, obtain the complete structure, is the typical characteristics of rotavirus, infectious virus genome, without liquid mutations, with a final concentration of 0.00925% formaldehyde inactivated, first at 56 C 30min heating at 37 DEG C, and then continue to 72h after incubation, three blind passages prove that there is no residual virulence, virus was completely inactivated. The quantitative detection of antigen, then inactivated rotavirus P[2]G3 and aluminum hydroxide adjuvant adsorption, and the product prepared by mixed reagent of hepatitis A vaccine.
The second part of the experiment: the evaluation of immunological effect.
Method: the experimental mice were divided into blank group, control group and experimental group. Then the evaluation of immune effect of vaccine in mice. The combined vaccine by intramuscular injection, each time interval of two weeks were immune, immune 3 times. Since the first two weeks after immunization were detected in serum anti RV antibodies and feces the secretion of antibodies, the changes of CD4+T lymphocyte and CD8+T lymphocyte immune analysis.
Results: second weeks after the first immunization, all mice anti RV antibody positive; two times at fourth weeks after immunization, anti HAV antibodies were detected in third; sixth weeks after immunization, the antibody level reached the peak of serum anti RV enzyme antibody titer was 80865.76 + 1953.97, RV antibody titers of anti secretory 4.54 + 0.58, neutralizing antibody titers of anti RV 10240, anti hav antibody titer of 6.53. cell immune reaction of CD4+T 28.50876 + to 14.52 + 3.83, CD8+T reaches 3.9 + 0.72. by t test, more than P0.05.. The experimental results show that the inactivated rotavirus and inactivated hepatitis A virus vaccine can induce specific anti RV anti HAV humoral immune response and humoral immune response, can also produce cellular immune reaction obviously. This study provides some scientific basis for the development of IRV and IHAV combined vaccine.

【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

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