Question

Describe a strategy for how you would make a vaccine against a virus using genetic engineering and protein expression and purification system (i.e. from gene cloning to protein purification).

Answer 1

Ans:- The following steps in the production of vaccines using various approaches.

Genetic engineering

In genetic engineering or cloning approach first we will identify the specific sequence of the virus that produces it's specific viral coat protein. Now, once we know the sequence of the outer coat protein, we will amplify these sequence from Genomic DNA or cDNA of virus ( depending upon if the virus is DNA virus or RNA virus) via PCR. These PCR of viral coat protein will be cloned in into a expression vector containing a tag like his tag for easy purification.

The arrangement of gene will be coat protein gene then protease site then his tag. Protease site is important because during the purification we can cut the his tag and get purified protein without any tag. Clone this expression vector into E.coli. once Clones are confirmed then we will move to protein expression.

Protein Expression

Cloned expression vector is transformed into suitable bacterial host. After, transformation the colony was incoculated in LB tubes and further induction of viral coat protein was done using IPTG. After, induction cell lysis was performed. Followed by SDS PAGE. SDS PAGE gel electrophoresis will tell whether your viral coat protein is produced or not. Once, the viral coat protein band appears in the gel means expression of protein is done. We will move to protein purification.

Protein Purification

Induced protein lysate were loaded onto the Ni- NTA column. As we know that nickel binds specifically to his tag present in your protein. So, once you load the lysate onto the column your viral coat protein will bind to the column and other will be washed away. After, a few round of washing to remove nonspecific protein. We will Elute the viral proteins using immidazole.

The purity of purification is again checked by SDS PAGE. After Purification add the specific protease which will only cleave away your tag not your protein. Further, cleaved protein is subjected to gel filtration where you Elute your viral proteins.

Now, your protein will be ready for vaccine against that specific virus from where you have isolated viral coat protein gene.

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