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عنوان:

کلون سازی و بیان نانوبادی دوگانه علیه گیرنده های MUC1 and CD3 در میزبان باکتریایی



گروه تخصصی:  پزشکی

سازمان مجری:  پژوهشکده سرطان پستان 

گروه پژوهشی: پروتئین های نوترکیب

پژوهشگران: 
اسمعیلی رضوان (مسئول طرح)
فرهمند لیلا (مسئول طرح)
مجیدزاده اردبیلی کیوان (همکار طرح)
مجیدانصاری علیرضا (همکار طرح)
صنعتی حسن (همکار طرح)
زارع علی اکبر (همکار طرح)
صالحی ملیحه (همکار طرح)

تاریخ خاتمه:  زمستان 1395

کارفرما: 

خروجی طرح: 
 
تلفن: 

نشانی سازمان مجری: 
 

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کلیدواژگان: نانوبادی دوگانه,CD3 و درمان هدفمند,MUC1

 
 
Title:

Cloning and expression of anti MUC1& CD3 bispecific nanobody in bacterial host



Abstract:

Introduction: Cancer therapy based on antibodies and their different classes were caused to appear outstanding clinical results in improvement of patients suffering. Nanobodies are the variable single domain of camel antibodies. These smallest antigen binding fragments have unique advantages compared to the conventional therapeutic antibodies. MUC1 is overexpressed in more than 90% of breast cancers and correlates with metastasis and recurrence. Bi-specific Nanobodies, not only bind to antigen of cancer cell but also can stimulate cytotoxic T cells. In addition, it will have a synergistic effect on tumor cell eradication and deals with tumor escape. Materials and Methods: In order to expression of nanobody in bacterial host, a gene cassette was designed that include the nanobody sequences of anti-CD3 and anti-MUC1. Two nanobodies were conjuncted by a IgG2 Lama hinge linker together. The aim of additional signal sequence at the beginning of construct was producing secreted proteins and histidine sequence due to purification was added at the end of construct. After cloning, construct was transformed into the bacterial host. Nanobody expression was done in bacteria and it was purified by chromatography. The ability of bispecific anti MUC1 nanobody produced by the bacterial host to inhibit the proliferation of human breast adenocarcinoma cell line SKBR3, which overexpresses MUC1, performed by MTT Cell Proliferation Assay. Also, immunomodulatory effect of anti-CD3 was assayed using Jurkat T cells, which overexpresses CD3 as well as performed for anti muc1 nanobody. Binding of anti MUC1× CD3 to the immunomodulatory effect cells SKBR3 and Jurkat T cells were determined by flow-cytometry. The abilities of anti MUC1 nanobody to induce apoptosis was measured via apoptosis detection kit. Results: Bi-specific anti MUC1×CD3 nanobody was designed successfully. All stages of cloning and expression of nanobody were done appropriately in E. coli. Evaluation of protein concentration after purification indicated high level expression of nanobody in the bacterial host. The binding of bispecific anti MUC1× CD3 nanobody to the MUC1 and CD3 receptor was tested by measuring the dose-dependent effects on the proliferation of SKBR3 and Jurkat T cells respectively. It has been shown that anti MUC1 nanobody produced in E. coli can induce the apoptosis of SKBR3 cells. Discussion: As compared to the other methods of producing antibody which are more expensive and time-consuming, bi-specific nanobody against MUC1 & CD3 may be more functional and effective in some host with economic cost. According to the individual characteristics of nanobodies, after passing more developmental steps, it can be used in diagnosis and targeted therapy of cancer patients.



Keyword(s): Bi-specific nanobody,MUC1,CD و 3 targeted therapy