【作者】 于舒洋；

【导师】 李宁； 梁米芳；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 肾综合征出血热(HFRS)是由布尼亚病毒科汉坦病毒属汉坦病毒引起的一种急性病。血清学调查证实在五大洲都有汉坦病毒疫源地存在的证据。利用单克隆抗体治疗HFRS具有常规疗法无可比拟的优势。为获得乳腺高效表达人源重组抗体(rHMAbs)的小鼠模型,分别构建抗体基因轻链表达载体pBC1-hG2L和重链表达载体pBC1-hG2H,等比例混合利用显微注射方法共同注射受精卵雄原核制备转基因小鼠。在75只小鼠,用PCR和Southern杂交的方法检测出6(2♀,4♂)只为双链整合转基因阳性小鼠和6只仅整合重链的阳性小鼠,没有获得只整合轻链转基因阳性小鼠。转基因整合率16%,双链整合率为8%。将双链整合的阳性小鼠与野生型小鼠配种扩群。Western Blot检测转基因阳性母鼠的乳汁中rHMAbs的表达,在F0代鼠(F0-5)和F1代鼠(F1-6、F1-9、F1-20、F1-28、F1-38、F1-64)全部检测到重组人HFRS抗体的表达。ELISA进一步定量分析表明全部检测的双链整合母鼠乳汁中重组人HFRS抗体的表达量均在1mg/ml以上,表达量最高的F1-6号小鼠达到6.6mg/ml。免疫荧光试验(IFA)实验检测结果表明转基因小鼠乳中rHMAbs具有与HNTV和SEOV两个亚型的汉坦病毒特异结合的活性,微量荧光减少法中和实验检测结果表明rHMAbs可以在一定的滴度下在体外中和具有活力的病毒使之失去侵染细胞的能力。将乳中rHMAbs活力较高转基因鼠再次与野生型小鼠配种,采取被转基因鼠乳哺育的转基因鼠后代乳鼠(5日龄)血清,分析rHMAbs通过哺乳后是否能够进入小鼠体内及其对小鼠的保护作用。Western blot, ELISA检测转基因后代乳鼠血清结果表明rHMAbs可以被后代乳鼠吸收,但是分子量大小发生了改变。免疫荧光试验(IFA)和中和试验检测后代乳鼠血清中的rHMAbs,发现尽管rHMAbs受到了后代乳鼠自身的修饰,但其结合特异性与中和病毒的活性没有改变。这也间接证明了rHMAbs可以通过母婴免疫保护后代小鼠不受汉坦病毒的侵染。进一步对rHMAbs被后代乳鼠自身修饰后的分子量改变作了部分分析验证,结果表明rHMAbs重链没有发生显著的改变,而轻链不但受到N-linked糖基化修饰,而且还可能存在其它多种方式的修饰。本实验利用pBC1载体上的调控元件构建了分别含有肾综合征出血热轻、重链基因的转基因乳腺特异性表达载体并建立了能够遗传稳定的转基因鼠系。检测到rHMAbs在转基因小鼠乳中的高效表达(最高达到6.6mg/ml)。验证了转基因小鼠乳中rHMAbs具有完全的天然活性。为进一步制备转基因家畜奠定了基础,同时验证了rHMAbs通过哺乳可以对后代小鼠提供保护,并发现rHMAbs受到了小鼠自身的修饰而发生了分子量的变化,但仍然保持了天然活性,我们也由此推测是否内源的抗体通过乳鼠消化道后也会受到类似的修饰,如果此推论得到证实,那么这种生理机制的生物学意义将很值得进一步深入研究与探讨。更多还原

【Abstract】 Hemorrhagic fever with renal syndrome (HFRS) is a virosis caused by Hantavirus(HV), which belonged to the genus Hantavirus of family Bunyaviridae. It spreads wildly in five continents except Oceania and Antarctica and is very severe in Asia with a high mortality, especially in China. Tow vectors (pBC1-hG2L & pBCl-hG2H) bearing the light and heavy chains were constructed respectively to generate the transgenic mice which highly expressed recombinant human monoclonal antibodies (rHMAbs) against hantaviruses. Purified DNAs were diluted to 3ng/μl in TE buffer at 1:1 ratio and comicroinjected into the pronuclei of fertilized Kunming White eggs. In the 75 mice were produced by pronuclear injection. Seven (2 females and 4 males) lines of transgenic mice were identified to contain both heavy and light gene by PCR (Fig 2) and southern blot. Five mice only contain heavy gene and none of mice only have light gene. The integration rate is 16% and double chains integration rate is 8%. Transgenic mice were mated with wild mice and offspring were screened for the transgenes as described above. High level of rHMAbs against hantaviruses was detected through Western Blot in the milk of founder hAHT5 and F1 (F1-6、 F1-9、 F1-20、 F1-28、 F1-38、 F1-64) females. ELISA results showed that the expression levels of rHMAbs in the milk of all FO and Fl positive females were all over 1 mg/ml and the highest expression level was up to 6.6mg/ml (Fl-6).The Immunofluorescence test results showed the rHMAbs could specifically bind with the HTNV and SEOV and the rHMAbs expressed in the transgenic milk was that anticipant. We can found that the antibodies in the milk and sera both could neutralize hantaviruses in the related dilution through chemiluminescent focus reduction neutralization tests.The transgene female mice with highly expressed rHMAbs against hantaviruses were mated again. When the pups of transgenic mice and non-transgenic controls were five days old, the sera were collected to analyze in the next experiments. The results of western blot and ELISA showed that rHMAbs was detected in the sera of pups which were milked by positive transgenic mice, no matter whether they were transgenic or not. The result of further analysis indicated the light chain of rHMAb absorbed by the pups was N-linked glycosylated modification at least. It is very interesting that the rHMAbs in the pups’ sera still hold full bioactivity against HTNV, though the light chain of the antibody absorbed by the pups suffered N-linked glycosylation and other modifications. The rHMAb in the milk of transgenic mice and in the sera of pups suckled by them have been detected to have full natural bioactivity against HTNV, so it can provide protection against virus-induced disease. In summary, the minigene sequences which encode the heavy chain and light chain of the antibody contain part of introns that can improve the expression level of the antibody in mice milk and were respectively ligated into a commercial expression vector pBC1. The milk secreted by transgenic female mice was detected and the expression level of the rHMAbs in one line was up to 6.6mg/ml. In addition, the antibody was detected in the sera of pups milked by transgenic females. The antibodies in the milk of transgenic mice and in the sera of their offspring were shown to have full natural activity to provide protection against HTNV-induced diseases. The pups of transgenic mice can acquire passive immunity to this disease via their mothers. This experiment also affords a good strategy to produce milkcontaining the neutralizing IgGs aginst other major human pathogens, and provides an efficient and high-yield means to produce rHMAbs for therapeutic purposes via the mammary gland of transgenic livestock. Additionally, more researches should be carried out to investigate the reasons why the molecular weight of antibody in the sera was changed, though the natural activity of rHMAb was still held. We can presume the endogenous IgG modified by other molecule when the antibody is absorbed by their pups through milked更多还原