Sunday, November 3, 2019

Technical summary Essay Example | Topics and Well Written Essays - 500 words

Technical summary - Essay Example Mouse-adapted H1N1 A/PR/8/34 strains were used for the study. Genomic RNA (full-length) was acquired by infecting MDCK cells; the procedure was done under biosafety level-2 conditions. pFBHAhisKDEL and pFBMelHAhis plasmids were used to produce the recombinant baculoviruses BacHAhisKDEL and BacMelHAhis using Bac-to-Bac baculovirus expression system. The instructions provided by the manufacturer of the system were strictly followed. Control used was baculovirus vector BacNI (this is a baculovirus, which has no foreign gene). There was propagation and amplification of the recombinant baculoviruses in Sf21 insect cells in order to attain infective titers of around 108 plaque-forming units (pfu) (Gomez-Casado 36). Sf21 insect cells and the insect larvae (Trichoplusia ni) were infected using recombinant baculovirus dilution to attain the amount of pfu per dose required for each selection. Total soluble and non-denatured proteins (TSNDPs) were obtained through homogenization; these proteins were from baculoviruses infected T. ni larvae (Gomez-Casado 36). The proteins were prepared using various techniques for western blot (WB) assays and protein size determination. Recombinant HAhisKDEL protein purification from the infected larvae was done using Co2+-based immobilized metal affinity chromatography (IMAC) resins (Gomez-Casado 37). The response of HA-specific IgG (immunoglobin G) was measured using ELISA tests. Inhibition tests of hemagglutination were done for each of the duplicate based on instruction from the World Organization for Animal Health. Female mice (6 to 8 weeks old) were immunized and tested for virus challenge. The first group of mice (4) was immunized with TSNDP extract (containi ng HAhisKDEL protein) from the infected larvae. The second group (4 mice) was immunized with purified HAhisKDEL protein. Control group (3 mice) were immunized using TSNDP extract

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