| AD is a kind of common degenerative disease in central nervous system(CNS). It is generally accepted that the extracellular deposition of Aβ42 in senile dementia is the mian cause which led to AD,Recent researches suggest that not only the production and aggregation of intraneuronal AP42 is the beginning of the neurodegeneration,but the accumulation of intraneuronal AP42 disrupt neural and synaptic function and ultimately lead to neuronal degeneration and dementia.Therefore,clearing intracellular Aβ42 is the key to cure AD. Intrabody of anti-Aβ42 is the most effective method to remove Aβ42.But there are still some problems as following:①The early construction of antibody such as whole antibody IgG, single chain antibodies,was effective after treatment,clinical symptoms and signs of significant improvement, but it also induced activation of immune cells causing side effects such as encephalitis and meningitis in reaction to its treatment significantly reduced the effect, so it still remains in the laboratory research stage;②macromolecules protein through the blood-brain barrier(BBB) difficult play a role, intracerebroventricular injection or intraperitoneal injection into the brain or other means is not only difficult to manipulate, poor efficacy, but because of the special nature of drug delivery, the patient is difficult to tolerate .Eeven through penetrating the BBB ,it is difficult to maintain the effective therapeutic concentration to play a neuroprotective effect because of its short half life was soon degraded, it is difficult to apply clinically.Intrabody is kind of engineering antibody which can express intracellularly and locate at centain subcellular fraciton combing with target molecule to develop its biology function. With the development of gene engineering,the intrabody as a means of high-profile gene therapy, inhibition of viral replication has been widely used, especially HIV-1 replication, tumor gene therapy in recent years has been gradually extended to the central nervous system disease, transplant rejection and autoimmune diseases and other fields.Intrabody keep the character of common antibody except for high affinity and specificy,there are other advantages as following:①Better intracellular stability than small interference peptide and short reverse-nucleotide which are easily degraded,only 100～300 peptide can effectively resist degrading of protease;②Intrabody containing certain signal peptide can locate at specified cytoplasmic,such as SEKDEL anchoring at the endoplasmic reticulum,NLS anchoring at nucleus;③the scFvs-SEKDEL small molecular weight can be subjected to the endoplasmic reticulum-Golgi RP degradation pathway in cells, avoiding the extracellular antibody caused by non-specific immune response.Therefore we design and construct the rAAV which express secreted intrabody in vivo targeting combined and clearing away from intraneuronal Aβ42 to achieve the purpose of diagnosis and treatment of AD.Construction is as following:①asing on GenBank providing the sequence monoclone antibody of Aβ42 (Potent number WO 2008/156621 A1), we utilizes variable light and heavy of CDRs(CDR1andCDR3) by coupling the two with special linker .Analysising and designing the primers with DNASIS software through double PCR to receive the target genes mAbA/B.Ligating the target genes with vector pGEM-Teasy and identifing the recombined plasmid with restriction enzymes, they were tested by Sanger single termination the sequence of the target DNA.②Ligating the target genes with vector pGEM-Teasy and identifing the recombined plasmid with restriction enzymes, they were tested by Sanger single termination the sequence of the target DNA.③Three plasmids by calcium phosphate precipitation method were transferred into 293 small cell, and we conserve the recombinant AAV vectors carrying NT4-TAT-His-mAbA/B, RT-PCR method for determi-nation of recombinant Titer.④Immune Chemical test 6×His expression in the Hela cell infecting by AAV- mAbA/B and empty virus to verify the expression of this fusion gene validation.⑤Immune Chemical test 6×His and Aβ42 expression in the Hela cell infecting by AAV-mAbA/B and Aβ42 virus to verify preliminarily the protective effection of intrabody.The results show that: Restriction enzyme EcoR I digested pGEM-T Easy / mAbA / B, showed the purpose fragment of 132bp or 141bp, consistenting with the theoretical value;the results of DNASIS sequencing analysis software were consistent with that reported in GenBank. Digesting the plasmid pssHG-CMV/NT4-TAT-His-mAbA/B with restriction enzyme BamHI and PamHI and agarose gel electrophoresis of recombinant plasmid, we obtained the gene NT4-TAT-His-mAbA / B fragment of about 400bp, consistentin-g with the theoretical value.Real-time quantitative RT-PCR determination of viral titer 3.4×109PFU/ml. Immunohistochemical staining detected in the experimental group Hela 6×His expression did not express the empty virus group. Confocal microscope, the rAAV-mAbA/B and Aβ42 were expressed, the red anti-Aβ42 antibody binding to Aβ42, green represents His antibody binding antibodies and small molecules, they are both the same part of the expression of Hela, and the cell group and the control antibody Hela cell morphology group rules, not the number of fundamental change, and Aβ42 harm group showed a large number of cell debris scattered in the field of vision, Hela cells significantly reduced the number of irregular shape, showing polygonal mutation. Intracellular antibodies that are small molecules inside the cell by binding with Aβ42 cells play a protective role.Conclusion:①Cloned the objective gene mAbA / B by PCR successfully.②Constructed the penetrating "NT4-TAT-His-mAbA / B "fusion gene successfully.③Constructed pssHG/ NT4-TAT-His-mAbA/B rAAV (recombinant adeno-associated virus) and the restructured of higher concentrations of virus packaging successful.④NT4-TAT-His-mAbA/B could be secreted by rAAV vector in the expression of Hela cells.⑤Recombinant carrying NT4-TAT-His-mAbA/B of the AAV, mAb A/B and high specific binding of Aβ42 to Aβ42 injury in Hela cells can improve the shape .
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