पुनः संयोजक आरबीडी आधारित सार्स के टीके के लिए प्रोटोकॉल: प्रोटीन तैयार करना, पशु टीकाकरण और निराकरण जांच
Summary
इस प्रोटोकॉल सार्स के खिलाफ रीकॉम्बीनैंट रिसेप्टर – बाध्यकारी डोमेन (आरबीडी) आधारित सबयूनिट टीके के अध्ययन के लिए एक सामान्य प्रक्रिया का वर्णन करता है. यह अभिकर्मक और आरबीडी 293T कोशिकाओं में प्रोटीन, आरबीडी और माउस सीरा के निराकरण की गतिविधि की स्थापना की एक सार्स pseudovirus निराकरण परख का उपयोग का पता लगाने के साथ चूहों के टीकाकरण की अभिव्यक्ति के लिए तरीके शामिल हैं.
Abstract
Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.
Protocol
Discussion
सेल घनत्व के एक महत्वपूर्ण कैल्शियम फास्फेट आधारित अभिकर्मक की प्रभावकारिता को प्रभावित कारक है. हमारे अनुभव में, कम से कम 70% कक्षों की confluency सबसे अच्छा परिणाम लाता है. इस प्रकार, क्रम में अभिकर्मक दक्षता में सुधार करने के लिए, यह सिफारिश की है कि सेल घनत्व confluency के 50-70% के आसपास में रखा जाना. कैल्शियम फॉस्फेट अभिकर्मक विधि आम तौर पर अन्य अभिकर्मक तरीकों, जैसे लिपिड 16 अभिकर्मक के साथ तुलना में कम कुशल हो सोचा है. हालांकि, इस प्रोटोकॉल में, हम एक संशोधित कैल्शियम फॉस्फेट अभिकर्मक तरीका है जिससे लगातार और धीमी गति से एक भंवर में अभिकर्मक समाधान के मिश्रण उचित आकार और आकार के साथ एक वेग के गठन सुनिश्चित करता है का इस्तेमाल किया है, इस प्रकार अभिकर्मक दक्षता में सुधार.
Divulgations
The authors have nothing to disclose.
Acknowledgements
इस अध्ययन के राष्ट्रीय स्वास्थ्य संस्थान (एनआईएच) (AI68002 RO1) के संयुक्त राज्य अमेरिका के द्वारा समर्थित किया गया था.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| NaCl | Sigma | S7653 | ||
| Na2HPO4*7H2O | Sigma | S2429 | ||
| HEPES | Sigma | H3375 | ||
| CaCl2*2H2O | Sigma | C5080 | ||
| DMEM | Invitrogen | 12430 | ||
| HI FBS | Invitrogen | 10438 | ||
| Penicillin/Streptomycin | Invitrogen | 15140 | ||
| OPTI-MEM I Medium | Invitrogen | 31985 | ||
| Protease inhibitor cocktail | Roche | 11836170001 | ||
| Ni-NTA Superflow | Qiagen | 30450 | ||
| Sigma adjuvant system | Sigma | S6322 | ||
| Luciferase assay system | Promega | E1501 | ||
| Luciferase cell culture lysis reagent (5X) | Promega | E1531 | ||
| Amicon Ultra – 15 | Millipore | UFC901024 | ||
| Microfluor 96-well plates | Thermo Scientific | 7905 | ||
| Ultra 384 luminometer | Tecan Systems |
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