| 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...
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