Kloning dan Uji Ekspresi Gen Pengkode Highly Conserved Region (HCR) Protein Spike SARS-CoV-2 pada Lactococcus lactis NZ3900.

Violita, Melinda and Dr. apt. Valentina Yurina, S.Si., M.Si. and apt. Oktavia Rahayu Adianingsih, S.Farm., M.Biomed. (2021) Kloning dan Uji Ekspresi Gen Pengkode Highly Conserved Region (HCR) Protein Spike SARS-CoV-2 pada Lactococcus lactis NZ3900. Sarjana thesis, Universitas Brawijaya.

Abstract

Pandemi COVID-19 yang disebabkan oleh SARS-CoV-2 pada akhir tahun 2019 telah menyebabkan masalah kesehatan masyarakat yang utama. Virus corona diketahui bermutasi dengan cepat, terutama pada protein spike. Vaksin merupakan upaya yang efektif untuk mencegah infeksi virus. Pada protein spike S2 terdapat highly conserved region (HCR) yang berpotensi sebagai kandidat vaksin karena dapat mengenali berbagai strain virus corona. Pengembangan vaksin menggunakan vektor bakteri hidup sebagai media penghantaran dapat menginduksi sistem imun mukosa yang sangat penting untuk pertahanan terhadap infeksi virus yang menyerang saluran pernapasan. Vaksinasi melalui rute mukosal sangat praktis dan tidak invasif. Hal ini berbeda dengan vaksinasi melalui rute injeksi yang berisifat invasif, sementara ada kecenderungan perlunya booster berulang untuk vaksinasi SARS-CoV-2. Penelitian ini bertujuan untuk melakukan kloning dan uji ekpresi protein spike S2 HCR dengan menggunakan Lactococcus lactis sebagai inang. Metode yang dilakukan yaitu gen pengkode HCR protein spike S2 disintesis secara kimia dan dikonstruksi dalam plasmid pNZ8149. Hasil konstruksi selanjutnya ditransformasikan ke L. lactis NZ3900 dengan menggunakan metode elektroporasi. Kemudian dilakukan induksi ekspresi protein spike S2 HCR menggunakan nisin. Selanjutnya dilakukan analisa ekspresi protein dengan metode SDS-PAGE, Western blotting, dan ELISA. Hasil penelitian menunjukkan protein HCR yang diekspresikan pada L. lactis NZ3900 menghasilkan pita pada ukuran ±20 kDa pada analisis SDS-PAGE dan Western blotting. Berdasarkan kuantifikasi dengan metode ELISA, konsentrasi nisin 40 ng/mL menunjukkan hasil tertinggi. Bakteri L. lactis NZ3900 yang membawa plasmid pNZ8149 rekombinan dapat mengekspresikan protein spike S2 HCR dengan induksi nisin. Hal ini dapat menjadi tahap awal pengembangan vaksin virus corona yang diberikan melalui rute mukosal

English Abstract

The COVID-19 pandemic caused by SARS-CoV-2 in late 2019 has caused major public health problems. Coronaviruses are known to mutate rapidly, especially in spike proteins. Vaccines are effective tools to prevent viral infections. The spike protein S2 contains a highly conserved region (HCR) which has potential as a vaccine candidate because it is able to recognize various strains of the coronavirus. The development of vaccines using live bacterial vectors as a delivery carrier can induce the mucosal immune system which is very important for defense against viral infections that attack the respiratory tract. Vaccination via the mucosal route is very practical and non-invasive. So in contrast to vaccination via the injection route which is invasive, and there is a tendency of needed repeated boosters for SARS-CoV-2 vaccination. This research aimed to clone and analyze the expression of the spike protein S2 HCR using Lactococcus lactis as a host. The method used was the gene encoding the HCR spike protein S2 was chemically synthesized and constructed in the plasmid pNZ8149. The construction results were then transformed into L. lactis NZ3900 using the electroporation method. Then, S2 HCR spike protein expression was induced using nisin. Furthermore, protein expression analysis was performed using SDS-PAGE, Western blotting, and ELISA methods. The results showed that the HCR protein expressed in L. lactis NZ3900 produced a band at ±20 kDa in SDS-PAGE and Western blotting analysis. Based on quantification by the ELISA method, the nisin concentration of 40 ng/mL showed the highest result. Thus, it was concluded that L. lactis NZ3900 bacteria carrying the recombinant plasmid pNZ8149 can express the spike protein S2 HCR by nisin induction. This could be the initial stage of developing a coronavirus vaccine via the mucosal route.

Item Type:	Thesis (Sarjana)
Identification Number:	0521060288
Uncontrolled Keywords:	COVID-19, Vaksin, Protein spike, Highly conserved region, Lactococcus lactis, COVID-19, Vaccines, Spike protein, Highly conserved region, Lactococcus lactis
Subjects:	600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.1 Drugs (materia medica)
Divisions:	Fakultas Kedokteran > Farmasi
Depositing User:	Budi Wahyono Wahyono
Date Deposited:	28 Jan 2022 07:06
Last Modified:	23 Feb 2022 02:02
URI:	http://repository.ub.ac.id/id/eprint/189110

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