Infants and young children are susceptible population for the hand, foot, and mouth disease (HFMD) and gastroenteritis caused by enterovirus 71 and rotaviruses, respectively. For this population, vaccine is considered the effective and economical measure against many viral diseases. And, the diseases caused by enterovirus 71 and rotaviruses is vaccines preventable diseases.
Our project’s aim is to develop bivalent oral vaccine against gut microflora. On the one hand, we intend to develop a bivalent vaccine containing the two viruses’ antigens. Specifically, VP1 of enterovirus 71 and VP7 of rotaviruses are the viral proteins constitutes the capsid, respectively. Both proteins are exposed to the surface and are accessible for vaccine targeted VP1 and VP7. Moreover, proteins mutations promote the capability of virus particle infecting host. On the other hand, in considering of the lasting-time of vaccine in intestinal tract, oral vaccine is a better choice compared to injection-based injection. Because oral vaccine could improve distribution and allow self-administration relative to traditional injection-based formulations. Importantly, oral vaccine improve safety to avoid the blood-born infection, easy manufacturing and administration. In addition, the proinflammatory cytokines interleukin-12 (IL-12) was used as mucosal adjuvant to further enhance the immunome response. And, we used alacto-N-biose/lacto-N-biose I-binding protein (GL-BP) to assist the antigen and IL-12 in expressing on the surface of Bifidobacterium membrane.
Enterovirus 71 (EV71), belongs to Picornaviridae family, is a pathogen of hand, foot, and mouth disease (HFMD) in young children under 5 years old. The EV71 is a single-stranded RNA virus coated capsid which consists of structural proteins (VP1-VP4). Among the four structural proteins, VP1 is an immunodomaint protein due to distributing in the capsid surface. The VP1 mutations allow to escape the host immune response. Thus, the vaccines were designed based on the VP1 region and phase III clinical trials of vaccines have been completed in China.
As far as EV71, it was first isolated and characterized from cases of neurological disease in California in 1969. EV71, causing hand, foot, and mouth disease with complications including encephalitis, aseptic meningitis, pulmonary edema or pulmonary hemorrhage, acute flaccid paralysis and myocarditis, has become a serious public health problem in many counties, including Singapore, Vietnam, China and Australia. In China, an outbreak of EV71 infected HFDM occurred with more than 1.7 million cases and caused 905 deaths in 2010. Vaccines is considered to be most important means of preventing and controlling the disease.
Rotaviruses are the most common pathogen of gastroenteritis among infants and young children. Rotavirus are members of the Reoviridae family and transmitted through the fecal-oral route. These viruses consist of multilayered, non-enveloped particles with double-stranded RNA. VP4 and VP7 are exposed on the surface of capsid. Thus, these proteins are considered as the antigen to induce the neutralizing antibodies against the Rotavirus. VP7 is a calcium-binding glycoprotein and 11 viruses bearing VP7 mutation from human have been isolated. In China, the mortality rate of rotavirus infections in the infants and children within 0~5 years old is 0.58% in 2008.
Galacto-N-biose/galacto-N-biose I-binding protein (GL-BP) belongs to the family of ATP-binding cassette-type transporter from Bifidobacterium. The GL-BP are anchored in the membrane of gram-positive bacteria by lipid. Further, Bifidobacterium is dominant intestinal flora in breastfed infants and is considered to improve the human health.
Interleukin-12 (IL-12) is a pro-inflammatory type-1 cytokine which bridges the innate resistance and adaptor immunity. Also, IL-12 has long been studied as a potential vaccine adjuvant to stimulate the immune. In a study reported that oral administration of IL-12 could induce immune responses.
Oral vaccine is defined as a vaccine made from attenuated pathogenic microorganisms such as sugar pills or liquid that can be easily administered orally, such as polio vaccine. It is cheap to prepare, convenient to administer and easy to promote, but requires cryopreservation and has a short validity period. It is contraindicated in immunodeficient and immunocompromised individuals. It can be interfered with by other enteric viruses and affect the immunization effect, etc.
Combination vaccines were developed to prevent a wider range of diseases while reducing the number of vaccine injections. The significance of this is not only to improve vaccine coverage and vaccination rates, reduce the physical and psychological pain caused to infants and caregivers by multiple injections, reduce difficulties in vaccine administration, and reduce vaccination and administration costs, but also to reduce the dosage of preservatives and to reduce adverse reactions to vaccines.
The inactivated EV71 vaccine is an innovative vaccine developed in China for the prevention of HFMD caused by EV71 infection and is available for the prevention of HFMD that was officially approved in 2016. The vaccine is use in infants and young children from 2 months to less than 3 years of age. For rotavirus vaccines, there are two live and WHO-licensed rotavirus vaccines, RotaTeq and Rotarix, are available. In addition, Lanzhou Lamb Rotavirus (LLR-85) vaccine was developed by Lanzhou Institute of Biological Products (LIBP) and licensed in China. This vaccine is used in the children under 5 years.
Firstly, we already have the plasmids including pMD19-vp1, pMD19-vp7, and GFP. The plasmids of GLBP and IL-12 were obtained from a gene synthesis company. Then, the four gene fragments vp1, vp7, GLBP, GFP amplified by PCR, and got the three large DNA fragment though overlap extension PCR, including VP7-VP1, GLBP-VP7-VP1-GFP, and GLBP-VP7-VP1-IL12-GFP. All the fragments were obtained by gel extraction and treated by the restriction enzymes digestion, further integrated into expression vector pET28a by DNA ligase. These recombinant plasmids were transformed into DH5α using hot-shock method. Following, the colonies contained recombinant plasmids were verified by colony PCR and further confirmed by Sanger sequencing. Next, the recombinant plasmids transformed into the competent cells BL21 to express the virus protein and interleukin. The bacteria were induced by difference concentration of IPTG to express the protein fused GFP which is able to monitor the protein expression. Finally, the protein expression and purity were analyzed by SDS-PAGE and fluorescence microscopy.
PCR amplification instrument is also known as PCR gene amplification instrument, PCR nucleic acid amplification instrument, Polymerase chain reaction nucleic acid amplification instrument, is the use of PCR (Polymerase chain reaction) technology for specific DNA amplification of an instrument equipment, is widely used in medical, biological laboratories. Examples include mapping to determine whether a test will show a genetic disease, diagnosis of infectious diseases, gene duplication, and paternity testing.
In this experiment, PCR amplification instrument used for gene fragment amplification and colony PCR.
Pipette often used in the laboratory to remove a small amount or trace of liquid, different specifications, different specifications of pipette matching with different sizes of the nozzle, different manufacturers produce slightly different shapes, but the working principle and operation method is basically the same. pipette is a precision instrument, use and storage should be careful, to avoid damage, avoid affecting its range.
In this experiment, we use pipette to move and take some solution, such as the solution of the vp7 or pET28a plasmid.
Electrophoresis apparatus is an instrument for electrophoresis analysis. It is generally composed of power supply, electrophoresis tank, detection unit and so on. The so-called electrophoresis, refers to the movement of charged particles in the electric field, different substances due to the different charge and molecular weight, so in the electric field movement speed is different, according to which can be qualitative or quantitative analysis of different substances, or a certain mixture for component analysis or a single component extraction preparation.
In this experiment, we can find the magic of the plasmid through this instrument, in which we observe the move different distance of the uncut and cut plasmids.
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In this experiment, it mainly detects the absorbance signal of the vp7-vp1 and other things. This was used in the experiment to test the concentration of plasmid, DNA fragment, and bacteria.
Centrifuge is an instrument and equipment that simulates the earth's gravity field produced by the centrifugal force of an object rotating around a fixed axis of rotation, so as to make the object do sedimentation motion, so as to separate the levels of different densities and molecules T in the substance.
In this experiment, it mainly separation of solid and liquid phases in colloidal solutions.
Ultrasonic-sonicator is to convert electrical energy into sound energy through the transducer, which becomes a dense small bubble through the liquid medium, and these small bubbles burst quickly, producing energy like a small bomb, so as to play the role of breaking cells and other substances.
In this experiment, we use it to crush E. coli cells, and when we beginning to use it, we need to wash probe three times to avoid contamination. Specifically, the cells were disrupted and obtained the cell debris and soluble protein in cell extracts.
