Question

Please be as discriptive as possible. (Break it down dummy style please) Regarding the posted article “Hepatitis E vaccine debuts: Success of Chinese biotech partnership raises hopes for prevention of overlooked diseases” (Nature 10/29/2012): The workers made a recombinant subunit vaccine in E. coli host cells. Hepatitis E virus is a non-enveloped virus that contains an RNA genome and icosahedral capsid. Describe a stepwise process that could be used to develop and produce the vaccine. You have purified hepatitis E virus genomic RNA as starting material. (side question: if it’s recombinant would cloning be involved why or why not?)

Answer 1

The first thing that is required for making the vaccine is determining the sequence of the Genomic RNA sequence which can act as an antigen or can produce a protein that is virulent that can be antigenic and can elicit the immune response in the host.

So first step is to determine the portion that can act as an antigen and elicit the immune response .

after that the whole genetic engineering or recombinant DNA technology comes into picture.

Now we will have to cut our desired gene using restriction endonucleases , and isolate the desired gene , after isolating the desired gene.

Now this desired gene or gene of interest, which can produce antigenic protein must be amplified to mant copies , a technique called as polymerase chain reaction is used to amplify the given fragment of DNA for many cycles to produce many copies of the gene of interest.

Vector selection and insertion of desired gene into the vector:

A suitable vector is required for the insertion of the the desired gene , The Vector can be usually bacterial plasmids , the vector is taken and cleaved using endonucleases at specific restriction sites , and the desired gene is inserted into the vector and ligated using DNA ligase enzyme , Now we have a recombinant DNA or recombinant gene.

TRANSFORMATION : This recombinant DNA must be sent into a particular HOST for the expression of the desired gene.in this case it is E.coli. so the E.coli cells are made competent enough to allow the uptake of the DNA .

SCREENING OF TRANSFORMED CELL: The transformed cells means the cells into which the DNA of interest has entered are called transformed cells

once we get the transformed cells then we are ready for growing them and allowing them to express the protein that we require.

Now we intially grow them in labscale fermentors to stabilize the growth conditions required for the growth of the organisms . Once the growth conditions like media , pH , oxygen , nutrients all are stabilized we move into large scale fermentors or BIOREACTORS this is called Scaling up or upstream process.

Down Stream Process: The bacteria are cultured in large scale fermentors for a particular duration , post which the cells are harvested and after the cells are harvestes the protein of our interest must be extracted , purified , quality tested .

for the extraction of the protein we rupture the cell walls so that the product is released outside the cell.

once the product is released outside them we use various down stream techniques like chromatography of different types to filter the protein of interest.

The protein obtained after protein purification through chromatography is sent for quality analysis . certain stabilizers are added to keep the protein stable.

Finally its packed and used as a vaccine.